Nucleotide sequences of shrimp beta-actin and actin promoters and their use in gentic transformation technology

ABSTRACT

The present invention relates to isolated actin and β-actin nucleic acid promoter molecules from shrimp; nucleic acid expression cassettes including actin and β-actin promoter molecules isolated from shrimp; and expression vectors, host cells, and transgenic animals transduced with the isolated actin and β-actin nucleic acid promoter. Also disclosed are methods for imparting to an animal resistance against a pathogen; regulating growth of an animal; increasing stress tolerance in an animal; and increasing cold tolerance in an animal that involve transforming an animal with a nucleic acid construct including the isolated actin and β-actin nucleic acid promoter molecules of the present invention. The present invention also relates to isolated nucleic acid molecules encoding for actin and β-actin proteins or polypeptides, as well as the proteins or polypeptides themselves.

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/336,603, filed Dec. 4, 2001.

This invention was developed with government funding throughNOAA/National Sea Grant Award Nos. NA36RG0507, NA86RG0041, andNA16RG2254. The U.S. Government may have certain rights.

FIELD OF THE INVENTION

The present invention relates to nucleotide sequences of shrimppromoters which can be used in the construction of genetictransformation vectors for introducing desirable foreign DNA(s) intocommercially important shellfish and crustaceans.

BACKGROUND OF THE INVENTION

Infectious diseases among shrimp have taken a devastating toll onaquaculture production. Among the most harmful pathogens are viruses,bacteria, and protozoans, with viruses posing the greatest threat toshrimp survival rates. Bacterial and fungal infections in shrimp canusually be controlled effectively by applying available chemicaltreatments to shrimp populations in hatchery ponds or tanks. However,there are currently no effective chemicals or antibiotics to treat viraldiseases. Other strategies used in handling shrimp disease problemsinclude immunostimulation, vaccination, quarantining, and environmentalmanagement. These strategies are generally targeted at three elements:pathogens, host, and environment. Boosting the shrimp's natural defensesystem against pathogens is a non-specific approach to combatingdisease, yet, does not improve the shrimp's ability to cope with futureoutbreaks of the same disease since shrimp and other invertebrates lacka memory immune response based on antibody production. The lack of basicinformation about shrimp immunology is also another impediment to thedevelopment of efficient strategies for combating viral diseases viatraditional methods.

Viral diseases are the most devastating problem facing shrimpaquaculture. The four major viruses, including white spot syndrome virus(WSSV), yellow head virus (YHV), Taura syndrome virus (TSV), andinfectious hypodermal and hematopoietic necrosis virus (IHHNV), pose thegreatest threat to penaeid shrimp farming worldwide. The IHHNV was firstdetected in Hawaii in 1981, causing up to 90% mortality in juvenileshrimp, Litopenaeus stylirostris (Lightner et al., “InfectiousHypodermal and Hematopoietic Necrosis, a Newly Recognized Virus Diseaseof Penaeid Shrimp,” J. Invert. Pathol. 42: 62-70 (1983)). This virus hassince been reported to infect most Litopenaeus species (which waspreviously known as the Penaeus species), including the Pacific whiteshrimp, L. vannamei and the blue shrimp, L. stylirostris, causingtremendous economic losses worldwide (Brock, “An Overview of Diseases ofCultured Crustaceans in the Asia Pacific Region,” in Fish HealthManagement in Asia-Pacific. Report on a Regional Study and Workshop onFish Disease and Fish Health Management, ADB Agriculture DepartmentReport Series No. 1. Network of Aquaculture Centres in Asia-Pacific.Bangkok, Thailand, pp. 347-395 (1991); Flegel, “Major Viral Diseases ofthe Black Tiger Prawn (Penaeus Monodon) in Thailand,” in NRIAInternational Workshop, New approaches to viral diseases of aquaticanimals, Kyoto, Japan. Jan. 21-24, 1997, National Research Institute ofAquaculture, Nansei, Mie 516-01, Japan pp. 167-189 (1997)). TSV hasinfected United States farms rearing Litopenaeus vannamei since 1992 andhas caused more than 2 billion dollars in damage to aquaculture farms(Brock, “An Overview of Taura Syndrome, an Important Disease of FarmedPenaeus Vannamei,” in C. L. Browndy and J. S. Hopkins, (eds.), SwimmingThrough Troubled Water. Proceedings of the Special Section on ShrimpFarming, Baton-Rouge, La.: World Aquaculture Society pp. 84-94 (1995);Lightner et al., “Risk of Spread of Penaeid Shrimp Viruses in theAmericas by the International Movement of Live and Frozen Shrimp,” Rev.Sci. Tech. 16(1):146-60 (1997)). In Hawaii, both TSV and IHHNVinfections in shrimp farms have been frequently reported since 1994(MacMillan, “Shrimp Diseases in Hawaii, USA”. UNIHI-SG-FS-96-02.University of Hawaii Sea Grant College Program, Honolulu (1996)).Controlling viral diseases clearly represents a great challenge as thereare currently no effective chemicals or antibiotics to treat viralinfection. The serious effects of viral disease outbreaks among culturedshrimp coupled with a decline in natural fisheries of healthy shrimp(Pullin et al., “Domestication of Crustaceans,” Asian Fisheries Sci.11(1):59-69 (1998)), have led to a critical demand for advancedbiotechnological applications.

The two major penaeid shrimp species cultured in the Americas, L.vannamei and L. Stylirostris, have differing susceptibilities to TSV andIHHNV. L. vannamei is more resistant to IHHNV, but susceptible to TSV,whereas L. stylirostris is innately resistant to TSV but highlysusceptible to IHHNV (Lightner et al., “Strategies for the Control ofViral Diseases of Shrimp in the Americas,” Fish Pathology 33:165-180(1998)). Despite the relative resistance of L. vannamei to IHHNV, runtdeformity syndrome (RDS) was still observable in this shrimp specieswhen exposed to IHHNV. Although these viral diseases may not becompletely fatal, the reduced growth rate resulting from viral-inducedRDS results in immense revenue losses for shrimp farmers each year.

Systematic genetic selection is known to enhance disease resistance in anumber of farmed plants and animals, including fish (Gjedrem et al.,“Genetic Variation in Susceptibility of Atlantic Salmon toFurunculosis,” Aquaculture 97:1-6 (1991)). However, the efficacy ofbreeding for disease resistance in penaeid shrimp is not wellestablished because of the paucity of information about relevant geneticparameters, such as phenotypic and genetic variation, heritability, andgenetic correlations between traits. In response to viral-diseaseproblems facing the shrimp farming industry, the U.S. Marine ShrimpFarming Program (USMSFP), with funding from USDA/CSREES, has developed aselective breeding program to enhance disease resistance and improvegrowth in L vannamei (Moss et al., “Breeding for Disease Resistance inPenaeid Shrimp: Experiences From the U.S. Marine Shrimp FarmingProgram,” In: Proceedings of the 1^(st) Latin American Shrimp FarmingCongress (D. E. Jory, ed.), Panama City, Panama, 9 pp. (1998); Argue etal., “Selective Breeding of Pacific White Shrimp (Litopenaeus Vannamei)for Growth and Resistance to Taura Syndrome Virus,” Aquaculture204:447-460 (2002)). Although high between-family variation in responseto TSV challenge was observed in all groups of shrimp tested,heritability estimates (h²) for TSV resistance were low (h²_(full-sib)=0.14). Heritability describes the percentage of phenotypicvariance that is inherited in a predictable manner and is used todetermine the potential response to selection (Tave, “Genetics for FishHatchery Managers,” 2nd ed., AVL New York, 415 pp (1993)). Estimates ofh² typically are low for fitness traits, such as disease resistance, andphenotypes with h²≦0.15 are difficult to improve by selection. Althoughthe development of TSV-resistant strains of L. vannamei have benefitedshrimp farmers, breeding for TSV resistance is not a panacea to thehealth problems plaguing the industry. Viruses can mutate, therebyrendering selectively bred shrimp incapable of defending themselvesagainst new strains of virus. Furthermore, TSV resistance could benegatively correlated with resistance to other pathogens. There is alsothe potential to produce shrimp that respond well in disease-challengetests used in breeding programs, but perform poorly when stocked incommercial ponds.

The use of molecular biology techniques to produce pathogen-resistantstrains of shrimp through genetic transformation technology isconsidered a highly promising strategy for control of shrimp viraldisease (Mialhe et al., “Future of Biotechnology-Based Control ofDisease in Marine Invertebrates,” Mol. Mar. Biol. And Biotechnol.4(4):275-83 (1995); Bachere et al., “Transgenic Crustaceans,” WorldAquaculture 28(4):51-5 (1997)). In the past decade, pathogen-resistanttransgenic animals and plants have been developed (Beachy, “VirusCross-Protection in Transgenic Plants,” in D. P. S. Verma, and R. B.Goldberg, (eds.), Plant Gene Research: Temporal and Spatial Regulationof Plant Genes, New York: Springer Verlag pp. 313-327 (1998); Kim etal., “Disease Resistance in Tobacco and Tomato Plants Transformed withthe Tomato Spotted Wilt Virus Nucleocapsid Gene,” Plant Dis. 78:615-21(1993); Sin, “Transgenic Fish,” Rev. Fish Biol. 7(4):417-41 (1997)), butuse of such technology has only just begun for shrimp research. Whilemethods for detecting viral disease in shrimp, including polymerasechain reaction (Dhar et al., “Detection and Quantification of InfectiousHypodermal and Hematopoietic Necrosis Virus (IHHNV) and White Spot Virus(WSV) of Shrimp by Real-Time Quantitative PCR and SYBR Chemistry,” J.Clin. Microbiol. 39:2835-2845 (2001); Tang et al., “Detection andQuantification of Infectious Hypodermal and Hematopoietic Necrosis Virusin Penaeid Shrimp by Real-Time PCR,” Dis. Aquat. Org. 44(2):79-85(2001)), light microscopy, and transmission electron microscopy (Nunanet al., “Reverse Transcription Polymerase Chain Reaction (RT-PCR) Usedfor the Detection of Taura Syndrome Virus (TSV) in ExperimentallyInfected Shrimp,” Dis. Aquatic. Org. 34:87-91 (1998); Goarant et al.,“Arbitrarily Primed PCR to Type Vibrio Spp. Pathogenic for Shrimp,”Appl. Environ. Microbiol. 65(3):1145-1151 (1999); Chen et al.,“Establishment of Cell Culture Systems from Penaeid Shrimp and TheirSusceptibility to White Spot Disease and Yellow Head Viruses,” Meth, inCell Sci. 21:199-206 (1999); Toullec, “Crustacean Primary Cell Culture:a Technical Approach,” Meth. in Cell Sci. 21:193-8 (1999);Sukhumsirichart et al., “Characterization and PCR Detection ofHepatopancreatic Parvovirus (HPV) from Penaeus Monodon in Thailand,”Dis. Aquat. Org. 38:1-10 (1999), are widely used, methods forcontrolling viral disease in shrimp are still in development. The firststudies on genetic transformation of marine molluscs and shrimp wereinitiated in 1988 in France at IFREMER, in the United States at theUniversity of Maryland Biotechnology Institute, and in Australia atCSIRO. A few studies on the introduction of foreign DNA into shrimpembryos via transfection methods have obtained preliminary datademonstrating transient expression of a reporter gene by heterologouspromoters (Gendreau et al., “Transient Expression of a LuciferaseReporter Gene After Ballistic Introduction Into Artemia franciscana(Crustacea) Embryos,” Aquaculture 133:199-205 (1995)). Recent advancesin gene transfer technology such as these hold immense potential fordeveloping transgenic shrimp harboring genes that convey viral diseaseresistance or enhance shrimp growth rates. Gene transfer technology thusrepresents a practical alternative to the lengthy and expensiveselective breeding process (Wolfus et al., “Application of theMicrosatellite Technique for Analyzing Genetic Diversity in ShrimpBreeding Programs,” Aquaculture 152:35-47 (1997)), and provides apowerful tool for revolutionizing not only shrimp aquaculture, but alsolivestock husbandry in general.

Construction of an effective expression vector is an important steptoward implementing the genetic transformation process in animals. Theexpression vector is generally composed of three elements: a promoter, atarget gene, and a region having transcriptional termination signals.Among these three components, a suitable promoter is the most importantelement for a successful gene transformation system. The promoterdetermines where, when, and under what conditions the target gene shouldbe turned on.

A suitable promoter that is appropriate for aquaculture and acceptableto consumers should ideally be derived from marine origin and should notpose any potential health hazards. Several fish gene promoters have beensuccessfully isolated and used to drive foreign gene expression(Jankowski et al., “The GC Box as a Silencer,” Biosci. Rep. 7:955-63(1987); Zafarullah et al., “Structure of the Rainbow TroutMetallothionein B Gene and Characterization of its Metal-ResponsiveRegion,” Mol. Cell. Biol. 8:4469-76 (1988); Liu et al., “Development ofExpression Vectors for Transgenic Fish,” Bio/Technoloy 8:1268-1272(1990b); Gong et al., “Functional Analysis and Temporal Expression ofPromoter Regions From Fish Antifreeze Protein Genes in TransgenicJapanese Medaka Embryos,” Mol. Mar. Biol. Biotechnol. 1(1):64-72 (1991);Du et al., “Growth Enhancement in Transgenic Atlantic Salmon by the Useof Fish Antifreeze/Growth Hormone Chimeric Gene Constructs,”Biotechnology 10:176-81 (1992); Gong et al., “Transgenic Fish inAquaculture and Developmental Biology,” Current Topic in Develop. Biol.30:175-213 (1995); Chen et al., “Transgenic Fish and Aquaculture,”Biotechnol. Apl. 13(1):50 (1996); Chan et al., “PCR Cloning andExpression of the Molt-Inhibiting Hormone Gene for the Crab (Charybdisferiatus),” Gene 224:23-33 (1998); Gong, “Zebrafish Expressed SequenceTags and Their Applications,” Meth. Cell Biol. (zebrafish volume)60:213-233 (1998); Ju et al., “Faithful Expression of Green FluorescentProtein (GFP) in Transgenic Zebrafish Embryos Under Control of ZebrafishGene Promoters,” Dev. Genet. 25(2):158-67 (1999); Yoshizaki et al.,“Germ Cell-Specific Expression of Green Fluorescent Protein inTransgenic Rainbow Trout Under Control of the Rainbow Trout Vasa-LikeGene Promoter,” Int. J. Dev. Biol. 44(3):323-6 (2000)). Other promotersused to date in transgenic marine fish include mouse metallothionein(McEvoy et al., “The Expression of a Foreign Gene in Salmon Embryos,”Aquaculture 68:27-37 (1988); Rahman et al., “Fish Transgene Expressionby Direct Injection Into Fish Muscle,” Mol. Mar. Biol. Biotechnol.1:286-289 (1992)), heat shock promoters (Bayer et al., “A TransgeneContaining lacZ is Expressed in Primary Sensory Neurons in Zebrafish,”Development 115:421-446 (1992); Krone, “Several Unique Hsp 90 Genes areExpressed During Embryonic Development of Zebrafish,” Presented atSymposium on Advances in Molecular Endocrinology of Fish, May 23-25,Toronto, Canada (1993)), chicken β-actin promoter (Lu et al.,“Integration and Germline Transmission of Human Growth Hormone Gene inMedaka (Oryzias latipes),” presented at Second International MarineBiotechnology Conference, 1991, Baltimore, Md. (1991); Inoue et al.,“Introduction, Expression, and Growth-Enhancing Effect of Rainbow TroutGrowth Hormone cDNA Fused to an Avian Chimeric Promoter in Rainbow Fry,”J. Mar. Biotechnol. 1:131-4 (1993)), carp β-actin promoter (Liu et al.,“Functional Analysis of Elements Affecting Expression of the β∃-ActinGene of Carp,” Mol. Cell Biol. 10:3432-3440 (1990); Rahman et al., “FishTransgene Expression by Direct Injection Into Fish Muscle,” Mol. Mar.Biol. Biotechnol. 1:286-289 (1992)), the antifreeze protein promoterfrom the ocean pout (Macrozoarces americanus) (Gong et al., “FunctionalAnalysis and Temporal Expression of Promoter Regions From FishAntifreeze Protein Genes in Transgenic Japanese Medaka Embryos,” Mol.Mar. Biol. Biotechnol. 1(1):64-72 (1991); Hew et al., “AntifreezeProtein Gene Transfer in Atlantic Salmon,” Presented at SecondInternational Marine Biotechnology Conference, 1991, Baltimore, Md.(1991); Du et al., “Growth Enhancement in Transgenic Atlantic Salmon bythe Use of Fish Antifreeze/Growth Hormone Chimeric Gene Constructs,”Biotechnology 10:176-81 (1992)), and the histone promoter from the trout(Muller et al., “Introducing Foreign Genes Into Fish Eggs WithElectroporated Sperm as a Carrier,” Mol. Mar. Biol. Biotechnol.1:276-281 (1992)). Unfortunately, these promoters have disadvantages,including inconsistent transgenic expression, potential toxicity due totheir viral origin, and association with metabolic poisons and/ortumor-inducing sequences, all of which will present major stumblingblocks toward attaining FDA approval for the commercial use oftransgenic animals. However, isolation and use of promoter genes fromcrustacean shrimp has not been reported. Thus, the tremendous potentialpresented by gene transfer technology has not yet been realized inshrimp aquaculture due to the lack of a constitutive, non-inducible, andnon-developmentally regulated promoter to efficiently drive theexpression of heterologous genes in shrimp and other marine animals.

The present invention is directed to overcoming these and otherdeficiencies in the art.

SUMMARY OF THE INVENTION

The present invention relates to an isolated β-actin nucleic acidpromoter molecule from shrimp having a nucleotide sequence comprisingone or more (GC)-rich regions (i.e., regions rich in G and C).

The present invention also relates to an isolated nucleic acid moleculeencoding β-actin from shrimp, where the nucleic acid molecule either 1)has a nucleotide sequence of SEQ ID NO: 2; or 2) encodes a proteinhaving SEQ ID NO: 3.

The present invention also relates to an isolated shrimp β-actin havingan amino acid sequence of SEQ ID NO: 3.

The present invention also relates to expression vectors, host cells,and transgenic animals transduced with the isolated β-actin nucleic acidpromoter molecule from shrimp, and methods for imparting to an animalresistance against a pathogen, regulating growth of an animal, andincreasing stress tolerance in an animal, that involve transforming ananimal with a nucleic acid construct including the isolated β-actinnucleic acid promoter molecule from shrimp having a nucleotide sequencecomprising one or more (GC)-rich regions.

The present invention also relates to an isolated actin nucleic acidpromoter molecule from shrimp having a nucleotide sequence comprising(CATA)-rich repeats and (CACA)-rich repeats.

Another aspect of the present invention is an isolated nucleic acidmolecule encoding actin from shrimp, wherein the nucleic acid moleculeeither 1) has a nucleotide sequence of SEQ ID NO: 5; or 2) encodes aprotein having SEQ ID NO: 6.

The present invention also relates to an isolated shrimp actin having anamino acid sequence of SEQ ID NO: 6.

The present invention also relates to expression vectors, host cells,and transgenic animal transduced with the isolated actin nucleic acidpromoter molecule from shrimp, and methods of imparting to an animalresistance against a pathogen, regulating growth of an animal, andincreasing stress tolerance in an animal, that involve transforming ananimal with a nucleic acid construct including the isolated actinnucleic acid promoter molecule from shrimp having a nucleotide sequencecomprising (CATA)-rich repeats and (CACA)-rich repeats.

Transgenic strains of animals with new and desirable genetic traits mayoffer great benefits in marine aquaculture. For example, control ofinfectious diseases and acceleration of growth rate, two of the mostimportant challenges facing commercial shrimp aquaculture today, may beanswered by the application of recombinant DNA technology to theseproblems. However, genetic engineering of shrimp and other crustaceansrequires a suitable promoter that, ideally, is constitutive,non-inducible, non-developmentally regulated, and derived from marineorigin so as not to pose any potential health hazards. The presentinvention provides such promoters, and uses advanced recombinant DNAtechnology to produce transgenic marine animals in which one or moredesirable DNA sequences can be introduced.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the shrimp β-actin gene and itspromoter. Numbers represent nucleotide base pairs. UR: untranslatedregion, ATG: translation start site, SP: signal peptide, MP: maturepeptide, TAA: translation stop site. Regulatory regions (TATA, CAAT,CArG boxes and GC-rich regions) are present 100-1100 bp upstream fromthe translation start site. Drawing is not to scale.

FIG. 2 is a schematic diagram of the shrimp skeletal muscle actin(“actin”) gene and its promoter. Numbers represent nucleotide basepairs. UR: untranslated region, ATG: translation start site, SP: signalpeptide, MP: mature peptide, TAA: translation stop site. Regulatoryregions (TATA and CAAT boxes, CACA-rich and CATA-rich regions) arepresent approximately 500-1350 bp upstream from the translation startsite. Drawing is not to scale.

FIGS. 3A-B show a comparison of marker gene expression efficiency inshrimp muscle. Marker EGFP is shown in FIG. 3A, compared to DsRed, shownin FIG. 3B.

FIG. 4 is an ethidium bromide/agarose gel (2%) electrophoresis analysisof RT-PCR products encoding the TSV-CP with four sets of gene-specificprimers. DNA fragments of 471-bp, 574-bp, 1020-bp, 1123-bp, 573-bp and770-bp are shown in Lanes 2, 3, 4, 5, 6, and 7, respectively. A DNA sizemarker is shown in Lane 1.

FIG. 5 is an ethidium bromide/agarose gel (2%) electrophoresis analysisof RT-PCR products. Lane 1:100 bp DNA molecular marker; Lane 2: primerpair #1; Lane 3: primer pair #2; Lane 4: primer pair #3; Lane 5: primerpair #4; Lane 6: primer pair #5; Lane 7: primer pair #6; Lane 8: primerpair #7; Lane 9: primer pair #8; Lane 10: cloned IHHNV DNA bandgenerated with primer pair #8 that includes the full length sequence ofthe IHHNV-coat protein; and Lane 11: 100 bp DNA molecular marker.

FIGS. 6A-C are expression vectors consisting of the chimeric shrimpβ-actin promoter, sense or antisense TSV-CP target gene, and reporterβ-galactosidase gene (or EGFP gene). FIG. 6A is the pβ-ActinP2-β-Galvector construct. FIG. 6B is pβ-ActinP2-TSV-CP-AS (471 bp) with theTSV-CP target gene in the antisense orientation vector. FIG. 6C showspβ-ActinP2-TSV-CP-S (471 bp), constructed with the TSV-CP target gene inthe sense orientation.

FIG. 7 is the plasmid map of vector pβ-ActinP2-TSV-CP-S.

FIG. 8 is the plasmid map of vector pβ-ActinP2-TSV-CP-AS.

FIG. 9 is the plasmid map of vector pβ-ActinP2-β-Gal.

FIG. 10 is the plasmid map of vector pβ-ActinP2-P26.

FIG. 11 is the plasmid map of vector pβ-ActinP3-EGFP.

FIG. 12 is the plasmid map of vector pβ-ActinP1-EGFP.

FIG. 13 is a graph comparing the efficiency of the shrimp, chicken, andhuman cytomegalovirus (CMV) promoters in expressing EGFP in shrimp.

FIGS. 14A-B are graphs comparing the efficiency of the shrimppβ-ActinP2-β-Gal vector of against control vectors using microinjectionand electroporation. FIG. 14A shows the efficiency of β-ActinP2 promoterin β-Gal expression at different pulse lengths of electroporation of A.franciscana embryos. FIG. 14B shows efficiency of β-ActinP2 promotercompared to the CMV promoter in β-Gal expression in microinjected A.franciscana embryos.

FIG. 15 is a graph comparing hatching of L. vannamei shrimp embryosfollowing transfection with various ratios of plasmid DNA/SuperFect

FIG. 16 is an ethidium bromide/agarose gel (2%) electrophoresis analysisof RT-PCR detection of target gene, TSV-CP (antisense), expression inelectroporated L. vannamei. Lane 1: molecular marker. Lane 2:experimental shrimp. Lane 3: shrimp electroporated with PBS. Lane 4:positive control.

FIG. 17 is an ethidium bromide/agarose gel (2%) electrophoresis analysisof RT-PCR detection of target gene, TSV-CP (sense), expression inmicroinjected L. vannamei. Lane 1: molecular marker. Lane 2:experimental shrimp. Lane 3: negative control shrimp. Lane 4: positivecontrol.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an isolated β-actin nucleic acidpromoter molecule from shrimp having a nucleotide sequence comprisingGC-rich regions. This promoter, isolated and cloned from the Pacificwhite shrimp, Litopenaeus vannamei, has a nucleotide sequence of SEQ IDNO: 1, as follows: aaaaggatct aggtgaagat cctttttgat aatctcatga 60ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca 120aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 180caccgctacc agcggtggtt gtttgccgga tcaagagcta ccaactcttt ttccgaagta 240actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc 300caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca 360gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta 420ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag 480cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt 540cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc 600acgagggagc ttccaggggg aaacgcctgg tatctttata gtctgtcggg tttcgccacc 660tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg 720ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct 780ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata 840ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc 900gcccaatacg caaactcgcc tctccccgcg cgttggccga ttcattaatg cagctggcac 960gacaggtttc ccaactggaa agcgggcagt gagcgcaacc caattaatgt gagttagctc 1020actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt 1080gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caagctcgaa 1140attaaccctc actaaaggga acaaaagctg gagctcccca ccgcggtggc ggccgctcta 1200gaactagtgg atcccccggg ctgcaggaat ttggcacgag ctgccgaatt cggcacgagc 1260ccgagaggaa gcagcacgta cgctcgtccg cccttttagt aaaacaacaa caacaag 1297This β-actin promoter of the present invention is a constitutive,non-inducible, and non-developmentally regulated promoter. It issuitable for inducing expression of a protein encoded by a nucleic acidmolecule operably associated with the promoter molecule in an expressionvector. As shown in FIG. 1, the shrimp β-actin promoter of the presentinvention contains regulatory elements including a TATA box, CarG box,and CAAT box. It is interesting to note that the TATA box is locatedbetween two highly GC-rich regions. While the TATA and CAAT boxes areconserved among nucleic acid promoter molecules, the GC-rich regionslocated at 676-688 and 1161-1176 in the shrimp β-actin promoter of thepresent invention are not common, and appear to be characteristic ofthis particular promoter.

In the shrimp, the β-actin promoter contains a complex array ofcis-acting regulatory elements required for accurate and efficientinitiation of transcription and for controlling expression of theβ-actin gene. Transcripts of the shrimp β-actin gene are found in mostof the major shrimp organs including the eyestalk, brain, heart, andhepatopancreas, suggesting that the shrimp β-actin is a cytoplasmic formof actin whose expression is constitutive, non-developmentallyregulated, and non-inducible, and thus should remain constant throughoutthe lifespan of the shrimp.

The present invention also relates to an isolated nucleic acid moleculeencoding β-actin from the Pacific white shrimp, Litopenaeus vannamei,where the nucleic acid molecule has a nucleotide sequence of SEQ ID NO:2, as follows: atgtgtgacg acgaagtagc cgccctggtt gtagacaatg 60 gctccggcatgtgcaaggcc ggcttcgctg gtgacgatgc accacgagct gtgttcccct 120 ccatcgtcggccgaccccgt catcagggtg tgatggtcgg catgggccag aaggactcgt 180 acgtcggcgacgaggcccag agcaagcgag gtatcctcac cctgaaatac cccatcgagc 240 acggcatcgtcaccaactgg gacgacatgg agaagatctg gcatcacact ttctacaacg 300 agctccgcgtggcccccgag gagcaccccg tcctgctgac cgaggctccc ctcaacccca 360 aggctaaccgcgagaagatg acacagatca tgttcgagac cttcaacacc cccgccatgt 420 acgtggccatccaggccgtg ctgtccctgt acgcctccgg ccgtaccacc ggtatcgtgc 480 tcgactccggcgacggcgtg tcccacaccg tgcccatcta cgagggatat gccctgcccc 540 acgccatcctgcgtctggac ttggccggcc gcgacctcac agactacctg atgaagatcc 600 tgacggagcgtggctacacc ttcacgacca ccgccgagcg agaaatcgtt cgtgacatca 660 aggagaaactgtgctacgtg gccctggact tcgagcagga gatgaccacc gctgcttcct 720 cctcctcgctggagaagtcc tacgagctcc ctgacggcca ggtgatcacc atcggcaacg 780 agaggttccgctgccccgag gccctgttcc agccctcatt cctgggcatg gaatcctgcg 840 gcatccacgagaccacctac aactccatca tgaagtgcga cgtggacatc cgtaaggacc 900 tgtacgccaacaccgtgctg tccggaggca ccaccatgta ccctggcatc gccgacagga 960 tgcagaaggaaatcactgcc ctcgctccct ccaccatgaa gatcaagatc atcgccccac 1020 ccgagcgcaagtactccgtg tggatcggcg gctccatcct ggcctcgctc tccaccttcc 1080 agcagatgtggatcagcaag caggagtacg acgagtctgg accatcaatt gttcacagga 1140 agtgcttctaattaacaaaa tgtacatgat ataggctaca ctttttacat ttaattattc 1200 cattaggataaggattatgt tatttaaaag gaataaattc atttctacaa aaaaaaaaaa 1249 aaaaaaaaa

The nucleic acid molecule having a nucleotide sequence of SEQ ID NO: 2encodes a β-actin polypeptide or protein of the present inventionisolated from Litopenaeus vannamei, which has a deduced amino acidsequence of SEQ ID NO: 3, as follows: Met Cys Asp Asp Glu Val Ala AlaLeu Val Val Asp   1               5                  10 Asn Gly Ser Gly         15 Met Cys Lys Ala Gly Phe Ala Gly Asp Asp Ala Pro             20                  25 Arg Ala Val Phe      30 Pro Ser IleVal Gly Arg Pro Arg His Gln Gly Val          35                  40 MetVal Gly Met  45 Gly Gln Lys Asp Ser Tyr Val Gly Asp Glu Ala Gln     50                  55                  60 Ser Lys Arg Gly Ile LeuThr Leu Lys Tyr Pro Ile Glu His Gly Ile 65                  70                  75 Val Thr Asn Trp             80 Asp Asp Met Glu Lys Ile Trp His His Thr Phe Tyr                 85                  90 Asn Glu Leu Arg          95 ValAla Pro Glu Glu His Pro Val Leu Leu Thr Glu            100                 105 Ala Pro Leu Asn     110 Pro Lys AlaAsn Arg Glu Lys Met Thr Gln Ile Met         115                 120 PheGlu Thr Phe 125 Asn Thr Pro Ala Met Tyr Val Ala Ile Gln Ala Val    130                 135                 140 Leu Ser Leu Tyr Ala SerGly Arg Thr Thr Gly Ile Val Leu Asp Ser145                 150                 155 Gly Asp Gly Val            160 Ser His Thr Val Pro Ile Tyr Glu Gly Tyr Ala Leu                165                 170 Pro His Ala Ile         175 LeuArg Leu Asp Leu Ala Gly Arg Asp Leu Thr Asp            180                 185 Tyr Leu Met Lys     190 Ile Leu ThrGlu Arg Gly Tyr Thr Phe Thr Thr Thr         195                 200 AlaGlu Arg Glu 205 Ile Val Arg Asp Ile Lys Glu Lys Leu Cys Tyr Val    210                 215                 220 Ala Leu Asp Phe Glu GlnGlu Met Thr Thr Ala Ala Ser Ser Ser Ser225                 230                 235 Leu Glu Lys Ser            240 Tyr Glu Leu Pro Asp Gly Gln Val Ile Thr Ile Gly                245                 250 Asn Glu Arg Phe         255 ArgCys Pro Glu Ala Leu Phe Gln Pro Ser Phe Leu            260                 265 Gly Met Glu Ser     270 Cys Gly IleHis Glu Thr Thr Tyr Asn Ser Ile Met         275                 280 LysCys Asp Val 285 Asp Ile Arg Lys Asp Leu Tyr Ala Asn Thr Val Leu    290                 295                 300 Ser Gly Gly Thr Thr MetTyr Pro Gly Ile Ala Asp Arg Met Gln Lys305                 310                 315 Glu Ile Thr Ala            320 Leu Ala Pro Ser Thr Met Lys Ile Lys Ile Ile Ala                325                 330 Pro Pro Glu Arg         335 LysTyr Ser Val Trp Ile Gly Gly Ser Ile Leu Ala            340                 345 Ser Leu Ser Thr     350 Phe Gln GlnMet Trp Ile Ser Lys Gln Glu Tyr Asp         355                 360 GluSer Gly Pro 365 Ser Ile Val His Arg Lys Cys Phe    370                 375The deduced polypeptide of the shrimp beta-actin consists of a 63-aminoacid signal peptide and a 313-amino acid mature polypeptide. This shrimpβ-actin exhibits 99% amino acid homology with rainbow trout(Oncorhynchus mykiss) β-actin, 98% homology with fruit fly (Drosophilamelanogaster) β-actin5C, and 98% homology with chicken (Gallus gallus)β-actin.

The present invention also relates to an isolated nucleic acid promotermolecule from shrimp skeletal muscle actin having a nucleotide sequenceCATA-rich repeats and CACA-rich repeats. This actin promoter, isolatedand cloned from the Pacific white shrimp, Litopenaeus vannamei, has anucleotide sequence of SEQ ID NO: 4, as follows: ggactcgatc tggccatccctcttggctcg atgtcgcatt 60 cttgggtagt agcgtagggc tagttcgcgg taagtctgtataaaagggtg cacctgcctc 120 taaccaggta gtcgtgtcaa ggctcaaacc cgggtaagtgcaacgtgaca caaagcgtgg 180 ctcaggtgcg gcgaaaaggt gcaagtgtgt gttgaggtgaaaataaaagt ggtgtggagg 240 attaagaagg ttgctggacg tcacttatca ttgtgtgaacatttagtatg gattataaat 300 cagttgcttt catatgtatg tgtatctaga agtacgtgtggtttatgcca ttgtgaaaga 360 ggtttaagag aaacagaaag taatggagat gtaattacttttagttgctg aatattgcaa 420 acggttactt gagcacgagg acatctttat acattgtataatcattaaca tccatttcag 480 tggttaaaat aataattata atagataaag gcagttataatagacattgt ctattaatga 540 gagtcaatca gatatgctaa aaggatatag gctatgcgtaattatattgc ttacggaaaa 600 ctatttgaaa gattcacaca aaagataata tctgaactatattggattat attgaatgtt 660 gaccccacat ttatatatat atatatgtat atgtatatatatgtatttat atacataaat 720 atacacatat tcatgtatat atatatgtat atatacatatatatttatat atatgcatat 780 atatatgtat atatatatat atacatatgc atattcatatatatgtatat atatacatat 840 acatatatat gtatatatat gcataaatat atgtatatgaatatatatat atatatacat 900 acatacatac atatatatat atatatatat atatatatatatatatatat atatatacac 960 acacacacat gcacgcacat atacacacac acaaacacacacacatatat atgtaaatgc 1020 atacatatgt gtacacggac acatatatac acatatatacatacacacac aacagataca 1080 tatgcatata catacataca catatatctc tctctatatacatatatatg tatatatgtg 1140 tatatactta tacataaaaa cacacatttg cgcacacacatacacaatat acgacgcata 1200 catatgcata taaacacaca caaacacaca tatatccatatctgtctatc aatgtacaca 1260 tacacatata tctatatcta tctattaatg cacacacacagagacacaga cacacacatt 1320 tatgtataat atatatatat atatatatat atatatatatatatatatat atacatatat 1380 atttatgtat atacatacag tatatacaca catacacacaaacacattta tctatatatc 1440 tttatctatc tatctgccta tcaatcaatc aatctatctatctatctatc tataatatct 1500 atctatctat ctatctatct atctatctat ctatatatatgtatgtatgt atgtatacac 1560 acacacacat aagcacacac acacacatac acacacacacacacatatat acacacacaa 1620 tctgcctatc aatcaatcag tatatatgta cacacacacatatatatgcg cagactcata 1680 attgcacata tacacacaca catatatata cagtatatatatatatatat atatatatat 1740 atatatatat atatatatat atgtatatat atatatactcatatatacat acacacacat 1800 acacgcacac acacacacac acatatacac atgtaagttcacaggtacac agtatatata 1860 cataaatttt ttctctgtct ttatctttat gtattaacaaatgtgtatgt atatatatcg 1920 tatatgtata tatggtatat cagattcata catatgggtatatctataaa taattaatca 1980 ctcgtgtttc gtagaatgtc tgtaaatagg cgcaagcatgcacactcact tttaaacgct 2040 tgtgtacacc tgcacgtatg cacaaaaggt gtatgtaattttacataaat actacaacag 2100 cacatacata tacatgataa tgtttgggta atctcagtaatacaaaggta tctatgaaca 2160 ttaatctcat tttcatttta tctcaacagt gtcctctgacaactcaccta tatcacaatc 2200This promoter is suitable for inducing expression of a protein encodedby a nucleic acid molecule operably associated with the promotermolecule in an expression vector. The shrimp actin promoter of thepresent invention, as shown in FIG. 2, contains the expectedpromoter-associated TATA and CAAT boxes approximately 500 base pairsupstream from the translation start site. A unique characteristic ofthis promoter are CACA-rich and CATA-rich regions located upstream fromthe TATA and CAAT boxes at 878-893, 1071-1078, 1538-1549, 1554-1567, and1570-1585.

Another aspect of the present invention is an isolated nucleic acidmolecule encoding a skeletal muscle actin protein or polypeptide fromshrimp, wherein the nucleic acid molecule has a nucleotide sequence ofSEQ ID NO: 5, as follows: atgtgtgacg acgaagactc gtgtgcgctc gtgtgcgaca 60atggctccgg tatggtcaag gccggattcg caggagacga cgcccctcgc gccgtcttcc 120catccatcgt tggtcgtgct cgtcaccagg gtgtgatggt cggtatgggt cagaaggacg 180cctacgttgg tgatgaggcc cagagcaaac gtggtatcct caccctcaag taccccattg 240agcacggtat catcaccaac tgggatgaca tggagaagat ctggtaccac accttctaca 300atgagctccg tgttgcccct gaagagtccc ccacacttct cactgaggct cccctcaacc 360ccaaggccaa ccgtgagaag atgactcaga tcatgttcga gtccttcagt ctccctgcca 420tgtatgtgac catccaggct gtgctttctc tgtacgcctc tggtcgtacc actggtcagg 480tttgcgactc tggtgatggt gtgacccaca tggtccccgt ttatgaaggt ttcgctcttc 540ctcatgctat ccttcgtgtt gatttagctg gtcgtgatct taccaactac ctgatgaaga 600tcatgactga gcgtggctac tccttcacta ccaccgctga acgtgaaatc gtccgtgaca 660tcaaggagaa gctttgctac atcgcccttg atttcgaagg tgagatgaac gtcgctgctg 720cttcctcctc cttggacaag tcctacgaac ttcctgacgg tcaggtcatc accatcggta 780acgagcgttt ccgtgctccc gaggctctgt tccagccttc cttccttggt atggaatctg 840ctggtgttca ggaaaccgtg cacagctcta tcatgaggtg cgacattgac atcaggaagg 900atctgttcgc caatattgtg ctctctggtg gtactactat gtaccctggt attgctgacc 960gcatgcagaa ggaaatcaca accttggctc cttccaccat caagatcatc gccccacccg 1020agcgcaagta ctccgtgtgg atcggcggct ccatcctggc ctcgctctcc accttccagc 1080agatgtggat cagcaagcag gagtacgacg agtctggacc atcaattgtt cacaggaagt 1140gcttctaaat gtaggagact gaaaactttt attacagttg ataataaaat ccaaaagcaa 1198aaaaaaaaaa aaaaaaaa

The nucleic acid molecule having a nucleotide sequence of SEQ ID NO: 5encodes an actin protein or polypeptide of the present inventionisolated from the Pacific white shrimp, Litopenaeus vannamei, which hasa deduced amino acid sequence of SEQ ID NO: 6, as follows Met Cys AspAsp Glu Asp Ser Cys Ala Leu Val Cys  1               5                  10 Asp Asn Gly Ser          15 GlyMet Val Lys Gly Gly Phe Ala Gly Asp Asp Ala             20                  25 Pro Arg Ala Val      30 Phe Pro SerIle Val Gly Arg Ala Arg His Gln Gly          35                  40 ValMet Val Gly  45 Met Gly Gln Lys Asp Ala Tyr Val Gly Asp Glu Ala     50                  55                  60 Gln Ser Lys Arg Gly IleLeu Thr Leu Lys Tyr Pro Ile Glu His Gly 65                  70                  75 Ile Ile Thr Asn             80 Trp Asp Asp Met Glu Lys Ile Trp Tyr His Thr Phe                 85                  90 Tyr Asn Glu Leu          95 ArgVal Ala Pro Glu Glu Ser Pro Thr Leu Leu Thr            100                 105 Glu Ala Pro Leu     110 Asn Pro LysAla Asn Arg Glu Lys Met Thr Gln Ile         115                 120 MetPhe Glu Ser 125 Phe Ser Leu Pro Ala Met Tyr Val Thr Ile Gln Ala    130                 135                 140 Val Leu Set Leu Tyr AlaSer Gly Arg Thr Thr Gly His Val Cys Asp145                 150                 155 Ser Gly Asp Gly            160 Val Thr His Met Val Pro Val Tyr Glu Gly Ile Ala                165                 170 Arg Pro His Ala         175 IleLeu Arg Val Asp Leu Ala Gly Arg Asp Leu Thr            180                 185 Asn Tyr Leu Net     190 Lys Ile MetThr Glu Arg Gly Tyr Ser Phe Thr Thr         195                 200 ThrAla Glu Arg 205 Glu Ile Val Arg Asp Ile Lys Glu Lys Leu Cys Tyr    210                 215                 220 Ile Ala Leu Asp Phe GluGly Glu Met Asn Val Ala Ala Ala Ser Ser225                 230                 235 Ser Leu Asp Lys            240 Ser Tyr Glu Leu Pro Asp Gly Gln Val Ile Thr Ile                245                 250 Gly Asn Glu Arg         255 PheArg Ala Pro Glu Ala Leu Phe Gln Pro Ser Phe            260                 265 Leu Gly Met Glu     270 Ser Ala GlyVal His Glu Thr Val His Ser Ser Ile         275                 280 MetArg Cys Asp 285 Ile Asp Ile Arg Lys Asp Leu Phe Ala Asn Ile Val    290                 295                 300 Leu Ser Gly Gly Thr ThrMet Tyr Pro Gly Ile Ala Asp Arg Met Gln305                 310                 315 Lys Glu Ile Thr            320 Thr Leu Ala Pro Ser Thr Ile Lys Ile Ile Ala Pro                325                 330 Pro Glu Arg Lys         335 TyrSer Val Cys Ile Gly Gly Ser Ile Leu Ala Ser            340                 345 Leu Ser Thr Phe     350 Gln Gln MetCys Ile Ser Lys Gln Glu Tyr Asp Glu         355                 360 SerGly Pro Ser 365 Ile Val His Arg Lys Cys Phe     370                 375The deduced polypeptide of the shrimp actin consists of a 64-amino acidsignal peptide and a 311-amino acid mature polypeptide. This shrimpactin exhibits 94% amino acid homology with the tiger prawn (Penaeusmonodon) actin, 93% homology with the rattail fish (Coryphaenoidesacrolepis) skeletal alpha actin type 2, and 93% homology with human(Homo sapiens) alpha actin of the cardiac muscle.

Also encompassed by the present invention are fragments and variants ofthe above nucleic acid molecules and the proteins or polypeptides theyencode. Fragments of a nucleic acid molecule of the present inventionmay be made, for example, synthetically, or by use of restriction enzymedigestion on an isolated nucleic acid molecule. Variants may be made bythe deletion or addition of amino acids that have minimal influence onthe properties, secondary structure and hydropathic nature of thepolypeptide. For example, a polypeptide may be conjugated to a signal(or leader) sequence at the N-terminal end of the protein whichco-translationally or post-translationally directs transfer of theprotein. The polypeptide may also be conjugated to a linker or othersequence for ease of synthesis, purification, or identification of thepolypeptide.

Another aspect of the present invention relates to a nucleic acidconstruct containing the shrimp nucleic acid promoters of the presentinvention. This involves incorporating a nucleic acid promoter moleculeof the present invention into host cells using conventional recombinantDNA technology. Generally, this involves inserting the nucleic acidmolecule into an expression vector to which the nucleic acid molecule isheterologous (i.e., not normally present). A vector is generallyconstructed to include a promoter, a nucleic acid molecule targeted fortranscription and/or expression, and a 3′ regulatory region havingsuitable transcriptional termination signals.

“Vector” is used herein to mean any genetic element, such as a plasmid,phage, transposon, cosmid, chromosome, virus, virion, etc., which iscapable of replication when associated with the proper control elements,and which is capable of transferring gene sequences between cells. Thus,the term includes cloning and expression vectors, as well as viralvectors, including adenoviral and retroviral vectors.

Exemplary vectors include, without limitation, the following: lambdavector system gt11, gt WES.tB, Charon 4, and plasmid vectors such aspBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339,pR290, pKC37, pKC101, SV 40, pBluescript II SK+/− or KS+/− (see“Stratagene Cloning Systems” Catalog (1993) from Stratagene, La Jolla,Calif., which is hereby incorporated by reference in its entirety), pQE,pIH821, pGEX pET series (see F. W. Studier et. al., “Use of T7 RNAPolymerase to Direct Expression of Cloned Genes,” Gene ExpressionTechnology Vol. 185 (1990), which is hereby incorporated by reference inits entirety), and any derivatives thereof. Recombinant genes may alsobe introduced into viruses, such as vaccinia virus. Recombinant virusescan be generated by transfection of plasmids into cells infected withvirus.

Transcription of a target nucleic acid molecule in such a construct isdependent upon the presence of a promoter, which is a DNA sequence thatdirects the binding of RNA polymerase and thereby promotes mRNAsynthesis. In this aspect of the present invention the promoter is theβ-actin nucleic acid promoter molecule of the present invention havingSEQ ID NO: 1. The β-actin and actin promoters of the present inventionare a constitutive, non-inducible, non-developmental promoters. Aconstitutive promoter is a promoter that directs expression of a genethroughout the development and life of an organism. The promoters of thepresent invention are suitable, therefore, linked in the nucleic acidconstruct of the present invention to one or more nucleic acid moleculesencoding a target protein or polypeptide of interest for whichconstitutive expression in the selected host is desired.

Any target nucleic acid molecule(s) of interest may be operably linkedto this promoter molecule in a suitable vector, such that the nucleicacid molecule is under the control of the promoter of the presentinvention, including but not limited to, nucleic acids encoding viralproteins, such as coat proteins; growth regulating proteins, andproteins relating to enhanced stress tolerance in hosts transformed withsuch nucleic acid molecules, including heat shock proteins forincreasing tolerance to cold-related stress.

Also present in the vector is a 3′ regulatory region containing suitabletranscription termination signals selected from among those which arecapable of providing correct transcription termination andpolyadenylation of mRNA for expression in the host cell of choice,operably linked to a nucleic acid molecule which encodes for a proteinor polypeptide of choice. Exemplary 3′ regulatory regions for thenucleic acid constructs of the present invention include, withoutlimitation, the nopaline synthase (“nos”) 3′ regulatory region (Fraley,et al., “Expression of Bacterial Genes in Plant Cells,” Proc. Nat'lAcad. Sci. USA 80(15):4803-4807 (1983), which is hereby incorporated byreference in its entirety) and the cauliflower mosaic virus (“CaMV”) 3′regulatory region (Odell, et al., “Identification of DNA SequencesRequired for Activity of the Cauliflower Mosaic Virus 35S Promoter,”Nature 313(6005):810-812 (1985), which is hereby incorporated byreference in its entirety). An example of a commonly-used 3′ regulatoryelement for expression of genes of interest in animal cells is the SV40polyadenylation signal derived from the SV40 virus. Virtually any 3′regulatory element known to be operable in the host cell of choice willsuffice for proper expression of the genes contained in the plasmids ofthe present invention.

Also suitable in the nucleic acid construct of the present invention isa reporter gene (marker gene) such as β-galactosidase, luciferase, orgreen fluorescent protein (GFP) or enhanced green fluorescent protein(EGFP) gene of the bioluminescent jelly fish, Aequorea victoria (Inoue,“Expression of Reporter Genes Introduced by Microinjection andElectroporation in Fish Embryos and Fry,” Mol. Mar. Biol. andBiotechnol. 1(4/5): 266-270 (1992); Boulo et al., “Transient Expressionof Luciferase Reporter Gene After Lipofection in Oyster (Crassostreagigas) Primary Cell Cultures,” Mol. Mar. Biol. Biotechnol. 5(3):167-74(1996); Guillen et al., “Reporter Genes for Transgenic FishExperiments,” Biotechnol. Apl. 13(4):279-283 (1996); Arnone et al.,“Green Fluorescent Protein in the Sea Urchin: New ExperimentalApproaches to Transcriptional Regulatory Analysis in Embryos andLarvae,” Development 124(22):4649-4659 (1997); Husebye et al., “AFunctional Study of the Salmon GnRH Promoter,” Mol. Mar. Biol.Biotechnol. 6(4):357-363 (1997); Joore et al., “Regulation of theZebrafish Goosecoid Promoter by Mesoderm Inducing Factors and Xwnt1,”Mech. Dev. 55:3-18 (1997), which are hereby incorporated by reference intheir entirety). A reporter gene is added to the nucleic acid constructof the present invention in order to evaluate the promoter's capacity toeffectively direct expression of the target nucleic acid. Expression ofthe reporter gene is a good indication of whether the target gene wasproperly introduced into the host organism. The expression of thereporter gene also serves as a marker, helping to identify the organsand tissues in which the promoter is capable of driving target nucleicacid expression (Watson et al., “New Tools for Studying Gene Function,”In: Recombinant DNA, New York: Scientific American Books, pp. 191-272(1992); Winkler et al., “Analysis of Heterologous and HomologousPromoters and Enhancers in vitro and in vivo by Gene Transfer IntoJapanese Medaka (Oryzias latipes) and xiphophorus,” Mol. Mar. Biol. andBiotechnol. 1 (4/5):326-337 (1992), which are hereby incorporated byreference in their entirety). Expression of the β-galactosidase gene canbe monitored easily via spectrophotometry and expression of the EGFPgene can be visualized directly in live, transparent, transgenic shrimpunder a fluorescence microscope (Amsterdam et al., “The AequoreaVictoria Green Fluorescent Protein Can be Used as a Reporter in LiveZebrafish Embryos,” Dev. Biol. 171(1):123-129 (1995); Kain et al.,“Green Fluorescent Protein as a Reporter of Gene Expression and ProteinLocalization,” Biotechniques 19(4):650-655 (1995); Burlage et al.,“Green Fluorescent Protein in the Sea Urchin: New ExperimentalApproaches to Transcriptional Regulatory Analysis in Embryos andLarvae,” Development 124(22):4649-4659 (1997); Hong et al., “Dynamics ofNontypical Apoptotic Morphological Changes Visualized by GreenFluorescent Protein in Living Cells with Infectious Pancreatic NecrosisVirus Infection,” J. Virol. 73(6):5056-63 (1999), which are herebyincorporated by reference in their entirety) or hand-held UV Lamp (ClareChemical Research).

The promoter molecule of the present invention, a nucleic acid moleculeencoding a protein or polypeptide of choice, a suitable 3′ regulatoryregion, and if desired, a reporter gene, are incorporated into avector-expression system of choice to prepare the nucleic acid constructof present invention using standard cloning procedures known in the art,such as described by Sambrook et al., Molecular Cloning: A LaboratoryManual, Third Edition, Cold Spring Harbor: Cold Spring Harbor LaboratoryPress, New York (2001), which is hereby incorporated by reference in itsentirety, and U.S. Pat. No. 4,237,224 to Cohen and Boyer, which ishereby incorporated by reference in its entirety, which describes theproduction of expression systems in the form of recombinant plasmidsusing restriction enzyme cleavage and ligation with DNA ligase. Theserecombinant plasmids are then introduced by means of transformation andreplicated in unicellular cultures including prokaryotic organisms andeukaryotic cells grown in tissue culture.

In one aspect of the present invention, a nucleic acid molecule encodinga protein of choice is inserted into a vector in the sense (i.e., 5′→3′)direction, such that the open reading frame is properly oriented for theexpression of the encoded protein under the control of a promoter ofchoice. Single or multiple nucleic acids may be ligated into anappropriate vector in this way, under the control of one of thepromoters of the present invention.

In another aspect of the present invention, a target nucleic acidencoding a protein of choice is inserted into the vector in an antisenseorientation (3′→5′). The use of antisense RNA to down-regulate theexpression of specific plant genes is well known (van der Krol et al.,“Antisense Genes in Plants: An Overview,” Gene 72:45-50 (1988); van derKrol et al., “Inhibition of Flower Pigmentation by Antisense CHS Genes:Promoter and Minimal Sequence Requirements for the Antisense Effect,”Plant Mol Biol 14(4):457-66 (1990); Mol et al., “Regulation of PlantGene Expression by Antisense RNA,” FEBS Lett 286:427-430 (1990); andSmith et al., Nature, 334:724-726 (1988); which are hereby incorporatedby reference in their entirety). Antisense nucleic acids are DNA or RNAmolecules that are complementary to at least a portion of a specificmRNA molecule (Weintraub, “Antisense RNA and DNA,” Scientific American262:40 (1990), which is hereby incorporated by reference in itsentirety). Antisense methodology takes advantage of the fact thatnucleic acids tend to pair with “complementary” sequences. Bycomplementary, it is meant that polynucleotides are capable ofbase-pairing according to the standard Watson-Crick rules. In the targetcell, the antisense nucleic acids hybridize to a target nucleic acid andinterfere with transcription, and/or RNA processing, transport,translation, and/or stability. The overall effect of such interferencewith the target nucleic acid function is the disruption of proteinexpression.

Accordingly, both antisense and sense forms of the nucleic acids of thepresent invention are suitable for use in the nucleic acid constructs ofthe invention. A single construct may contain both sense and antisenseforms of one or more desired nucleic acids encoding a protein.

Alternatively, the nucleic acid construct of the present invention maybe configured so that the DNA molecule encodes an mRNA which is nottranslatable, i.e., does not result in the production of a protein orpolypeptide. This is achieved, for example, by introducing into thedesired nucleic acid sequence of the present invention one or morepremature stop codons, adding one or more bases (except multiples of 3bases) to displace the reading frame, and removing the translationinitiation codon (U.S. Pat. No. 5,583,021 to Dougherty et al., which ishereby incorporated by reference in its entirety). This can involve theuse of a primer to which a stop codon, such as TAA or TGA, is insertedinto the sense (or “forward”) PCR-primer for amplification of the fillnucleic acid, between the 5′ end of that primer, which corresponds tothe appropriate restriction enzyme site of the vector into which thenucleic acid is to be inserted, and the 3′ end of the primer, whichcorresponds to the 5′ sequence of the enzyme-encoding nucleic acid.

Genes can be effective as silencers in the non-translatable antisenseforms, as well as in the non-translatable sense form (Baulcombe, D. C.,“Mechanisms of Pathogen-Derived Resistance to Viruses in TransgenicPlants,” Plant Cell 8:1833-44 (1996); Dougherty et al., “Transgenes andGene Suppression: Telling us Something New?” Current Opinion in CellBiology 7:399-05 (1995); Lomonossoff, G. P., “Pathogen-DerivedResistance to Plant Viruses,” Ann. Rev. Phytopathol. 33:323-43 (1995),which are hereby incorporated by reference in their entirety).Accordingly, one aspect of the present invention involves nucleic acidconstructs which contain one or more of the nucleic acid molecules ofthe present invention as a nucleic acid which encodes a non-translatablemRNA, that nucleic acid molecule being inserted into the construct ineither the sense or antisense orientation. Several vectors have beenconstructed using the β-actin nucleic acid promoter of the presentinvention, as detailed below in the Examples.

Once the nucleic acid construct of the present invention has beenprepared, it is ready to be incorporated into a host cell. Accordingly,another aspect of the present invention relates to a recombinant cell,or “hos” cell containing a nucleic acid construct of the presentinvention. A variety of vector-host systems known in the art may beutilized to express the protein-encoding sequence(s). Primarily, thevector system must be compatible with the host cell used. Host-vectorsystems include, but are not limited to, the following: bacteriatransformed with bacteriophage DNA, plasmid DNA, or cosmid DNA;microorganisms such as yeast containing yeast vectors; mammalian cellsystems infected with virus (e.g., vaccinia virus, adenovirus, etc.);insect cell systems infected with virus (e.g., baculovirus); and animalcells, including marine fish, crustacean, particularly shrimp, and othermarine animals, infected by bacterial vector. Host cells are prepared bydelivery of vector into the host organism.

Three common methods of vector-expression for foreign nucleic aciddelivery are electroporation (Muller et al., “Introducing Foreign GenesInto Fish Eggs With Electroporated Sperm as a Carrier,” Mol. Mar. Biol.Biotechnol. 1:276-281 (1992); Powers et al., “Electroporation: a Methodfor Transferring Genes Into the Gametes of Zebra Fish (Brachydaniorerio), Channel Catfish (Ictalurus punctatus), and Common Carp (Cyprimuscario),” Mol. Mar. Biol. Biotechnol. 1:301-308 (1992); Sin et al., “GeneTransfer in Chinook Salmon by Electroporating Sperm in the Presence ofPRSV-lacZ DNA,” Aquaculture 117:57-69 (1993); Powers et al.,“Electroporation as an Effective Means of Introducing DNA Into Abalone(Haliotis rufescens) Embryos,” Mol. Mar. Biol. Biotechnol. 4(4):369-375(1995); Tsai et al., “Sperm as a Carrier to Introduce an Exogenous DNAFragment Into the Oocyte of Japanese Abalone (Haliotis divorsicolorsuportexta),” Transgenic Res. 6(1):85-95 (1997); Fraga et al.,“Introducing Antisense Oligonucleotides into Paramecium viaElectroporation,” J. Eukaryot. Microbiol. 45(6):582-8 (1998); Preston etal., “Delivery of DNA to Early Embryos of the Kuruma Prawn, Penaeusjaponicus,” Aquaculture 181:225-234 (2000), which are herebyincorporated by reference in their entirety), ballistic bombardment(Zelenin et al., “The Delivery of Foreign Genes Into Fertilized EggsUsing High-Velocity Microprojectiles,” FEBS Lett. 287(1-2):118-120(1991); Akasaka et al., “Introduction of DNA Into Sea Urchin Eggs byParticle Gun,” Mol. Mar. Biol. Biotechnol. 4(3):255-261 (1995); Gendreauet al., “Transient Expression of a Luciferase Reporter Gene AfterBallistic Introduction Into Artemia Franciscana (Crustacea) Embryos,”Aquaculture 133:199-205 (1995); Baum et al., “Improved BallisticTransient Transformation Conditions for Tomato Fruit AllowIdentification of Organ-Specific Contributions of I-Box and G-Box to theRBCS2 Promoter Activity,” Plant J. 12(2):463-9 (1997); Udvardi et al.,“Uptake of Exogenous DNA Via the Skin,” J. Mol. Med. 77(10):744-50(1999), which are hereby incorporated by reference in their entirety)and microinjection (Udvardi et al., “Uptake of Exogenous DNA Via theSkin,” J. Mol. Med. 77(10):744-50 (1999); Penman et al., “Patterns ofTransgene Inheritance in Rainbow Trout (Oncorhynchus Mykiss),” Mol.Reprod. Dev. 30:201-206 (1991); Damen et al., “TranscriptionalRegulation of Tubulin Gene Expression in Differentiating TrochoblastsDuring Early Development of Patella Vulgata,” Development 120:2835-2845(1994); Gaiano et al., “Highly Efficient Germ-Line Transmission ofProviral Insertions,” Proc. Natl. Acad. Sci. USA 93:7777-7782 (1996);Cadoret et al., “Microinjection of Bivalve Eggs: Application inGenetics,” Mol. Mar. Biol. Biotechnol. 6(1):7277 (1997); Li et al.,“Transfer of Foreign Gene to Giant Freshwater Prawn (Macrobrachiumrosenbergii) by Spermatophore-Microinjection,” Mol. Rerod. Dev.56(2):149-54 (2000), which are hereby incorporated by reference in theirentirety). Among these three methods, microinjection is considered to bethe most tedious, but most efficient, method for transferring foreignnucleic acid into marine and fresh water species. It allows precision indelivery of exogenous nucleic acid and increases the chances that atreated egg will be transformed. The introduced nucleic acid isultimately integrated into the chromosomes of the microinjectedorganism. Preston et al., “Delivery of DNA to Early Embryos of theKuruma Prawn, Penaeus japonicus,” Aquaculture 181:225-234 (2000) (whichis hereby incorporated by reference in its entirety), examined therelative efficiency of microinjection, electroporation, and particlebombardment for introducing nucleic acid into the embryos of the Kurumaprawn, Litopenaeus japonicus and found that microinjection is the mostreliable technique but very time consuming. Electroporation is adesirable method for large scale gene transfer, however, if the hostmortality is high, an alternative non-surgical technique (e.g.,spermatophore-microinjection), can be used as the delivery system. Whilestable expression is generally preferable, transient expression issuitable for some uses of the nucleic acid constructs of the presentinvention, therefore, the choice of delivery system in this aspect ofthe invention may vary depending on the type of expression desired.

After transformation, the transformed host cells can be selected andexpanded in suitable culture. Preferably, transformed cells are firstidentified using a selection marker simultaneously introduced into thehost cells along with the nucleic acid construct of the presentinvention. Suitable markers include those genes described above asreporter genes, i.e., β-glucuronidase, luciferase, EGFP, oradditionally, markers encoding for antibiotic resistance, such as thenptII gene which confers kanamycin resistance (Fraley, et al.,“Expression of Bacterial Genes in Plant Cells,” Proc. Nat'l Acad. Sci.USA 80(15):4803-4807 (1983), which is hereby incorporated by referencein its entirety), or gentamycin, G418, ampicillin, streptomycin,spectinomycin, tetracycline, chloramphenicol, and the like. A number ofantibiotic-resistance markers are known in the art and others arecontinually being identified. Any known antibiotic-resistance marker canbe used to transform and select transformed host cells in accordancewith the present invention. Cells or tissues are grown on a selectionmedium containing an antibiotic, whereby generally only thosetransformants expressing the antibiotic resistance marker continue togrow. Similarly, enzymes providing for production of a compoundidentifiable by luminescence, such as luciferase, are useful. Theselection marker employed will depend on the target species; for certaintarget species, different antibiotics, or biosynthesis selection markersare preferred.

The present invention also relates to a transgenic animal transformedwith a nucleic acid construct of the present invention described abovehaving a nucleic acid molecule encoding a protein under the control ofthe β-actin or actin promoter of the present invention. This involvespreparing a nucleic acid construct as described above containing theβ-actin or actin promoter, a nucleic acid molecule encoding a desiredprotein, and a 3′ regulatory region for termination, incorporating thenucleic acid construct into a suitable vector-host system, andtransforming an animal using a suitable delivery system, such as thosedescribed above. Animals suitable for this aspect of the presentinvention include, without limitation, marine fish; crustaceans,including shrimp and prawns; shellfish; and insects.

When stable transformation of a transgenic animal is achieved, the geneis incorporated into the organism's genome, and the gene is, therefore,heritable. Accordingly, the present invention also relates to theprogeny of the a transgenic animal transformed with the nucleic acidconstruct described above having a nucleic acid molecule encoding aprotein under the control of the β-actin or actin promoter of thepresent invention, wherein the progeny harbors the transformed nucleicacid.

Another aspect of the present invention is a nucleic acid expressioncassette including a β-actin promoter molecule isolated from shrimphaving SEQ ID NO: 1; a multiple cloning site; an operable terminationsegment; and a nucleic acid molecule encoding a detectable marker. Inthis aspect, a nucleic acid expression cassette is prepared generally asdescribed for the making of the nucleic acid construct having theβ-actin promoter of the present invention, with the promoter moleculeand a suitable 3′ termination segment (meaning a polyadenylation signaland a termination signal). However, the promoter is incorporated into avector having a multiple cloning site (MCS) for the insertion of one ormore nucleic acid molecules of choice by a user. In one embodiment theexpression cassette also contains a detectable marker. Exemplary markersinclude, without limitation, those named above. The promoter molecule, asuitable 3′ termination segment, and, if desired, a detectable marker,are ligated into a vector having a MCS, using standard cloningprocedures known in the art, such as described by Sambrook et al.,Molecular Cloning: A Laboratory Manual, Third Edition, Cold SpringHarbor: Cold Spring Harbor Laboratory Press, New York (2001), and U.S.Pat. No. 4,237,224 to Cohen and Boyer, which are hereby incorporated byreference in their entirety.

The present invention also relates to a method of imparting to an animalresistance against a pathogen. This involves transforming an animal withthe nucleic acid construct of the present invention described abovehaving the actin or β-actin promoter of the present invention, a nucleicacid molecule encoding a protein for resistance to a pathogen, and anoperable 3′ regulatory region. In one aspect of the present invention,the pathogen is a virus. Exemplary viruses against which resistance isimparted include those selected from the group consisting of white spotsyndrome virus (WSSV), yellow head virus (YHV), Taura syndrome virus(TSV), and infectious hypodermal and hematopoietic necrosis virus(IHHNV). In one embodiment of the present invention, the nucleic acidmolecule encodes a viral coat protein, or a fragment thereof. Suitablenucleic acid molecules are those encoding for the viral coat protein orpolypeptide of (WSSV), (YHV), (TSV), and (IHHNV). One or more coatprotein-encoding nucleic acid molecules can be used in a singleconstruct, so as to confer resistance to multiple viruses to one animalwith a single vector.

While not wishing to be bound by theory, by use of the constructs of thepresent invention, it is believed that viral resistance transgenicanimals can result using RNA-mediated post-transcriptional genesilencing. The strategy is to introduce a transgene consisting of senseand/or antisense versions of target gene (for examples, TSV coat proteinand the IHHNV coat protein) fragments into a host animal, so that theexpressed RNA transcripts will interfere with the translation process ofthe TSV and IHV coat protein genes, thereby inhibiting viral replicationin the animal.

More particularly, the silencer DNA molecule is believed to boost thelevel of heterologous RNA within the cell above a threshold level. Thisactivates the degradation mechanism by which viral resistance isachieved.

Posttranscriptional gene silencing (PTGS) based on RNA interference(RNAi) destroys RNA in a sequence-specific manner (Baulcombe, “RNASilencing,” Curr. Biol. 12(3):R82-4 (2002); Hutvagner et al., “RNAi:Nature Abhors a Double-Strand,” Curr. Opin. Genet. Dev. 12(2):225-232(2002), Hutvagner et al., “A MicroRNA in a Multiple-Turnover RNAi EnzymeComplex,” Science 297(5589):2056-2060 (2002), which are herebyincorporated by reference in their entirety) and functions in thenatural immunity of animal cells. Significant progress in the area ofviral resistance through RNA-mediated gene silencing has been achievedthrough research of RNAi in plants (Waterhouse et al., “Virus Resistanceand Gene Silencing in Plants Can be Induced by Simultaneous Expressionof Sense and Antisense RNA,” Proc. Natl. Acad. Sci. USA 95(23):13959-64(1998); Pang et al., “Resistance to Squash Mosaic Comovirus inTransgenic Squash Plants Expressing its Coat Protein Genes,” Mol. Breed.6:87-93 (2000); Vance et al., “RNA Silencing in Plants—Defense andCounterdefense,” Science 292(5525):2277-2280 (2001); Hongwei et al.,“Induction and Suppression of RNA Silencing by Animal Virus,” Science296:1319-1321 (2002), which are hereby incorporated by reference intheir entirety) and animals (Takayama et al., “Antisense RNA-MediatedInhibition of Viral Infection in Tissue Culture and Transgenic Mice,”In: Molecular Biology of RNA, Less, (ed.), pp. 299-310. New York (1989);Kim et al., “Examination of Antisense RNA and Oligodeoxynucleotides asPotential Inhibitors of Avian Leukosis Virus Replication in RP30 Cells,”Poultry Sci. 77:1400-10 (1998); Player et al., “Potent Inhibition ofRespiratory Syncytial Virus Replication Using a 2-5A-Antisense Chimeratargeted to Signals Within the Virus Genomic RNA,” Proc. Natl. Acad.Sci. USA 95:8874-9 (1998); Knight et al., “A Role for the RNase IIIEnzyme DCR-1 in RNA Interference and Germ Line Development inCaenorhabditis Elegans,” Science 293(5538):2269-2271 (2001); Tang etal., “Detection and Quantification of Infectious Hypodermal andHematopoietic Necrosis Virus in Penaeid Shrimp by Real-Time PCR,” Dis.Aquat. Org. 44(2):79-85 (2001); Korneev et al., “Suppression of NitricOxide (NO)-Dependent Behavior by Double-Stranded RNA-Mediated Silencingof a Neuronal NO Synthase Gene,” J. Neurosci. 22(11):RC227 (2002);Gitlin et al., “Short Interfering RNA Confers Intracellular AntiviralImmunity in Human Cells,” Nature, Online publication (2002), which arehereby incorporated by reference in their entirety). Current review ofRNA-mediated gene silencing mechanisms have been extensively described(Ahlquist, “RNA-Dependent RNA Polymerases, Viruses, and RNA Silencing,”Science 296:1270-1273 (2002); Plasterk, 2002, which are herebyincorporated by reference in their entirety). Examples of transgenicanimals include: inhibition of Moloney murine leukemia virus in micewith anti-sense RNA against the retroviral packaging sequences (Han etal., “Inhibition of Moloney Murine Leukemia Virus-Induced Leukemia inTransgenic Mice Expressing Antisense RNA Complementary to the RetroviralPackaging Sequences,” Proc. Natl. Acad. Sci. USA 88:4313-17 (1991),which is hereby incorporated by reference in its entirety), transgenicmice resistant to hepatitis virus (Sasaki et al., “Transgenic Mice WithAntisense RNA Against the Nucleocapsid Protein mRNA of Mouse HepatitisVirus,” J. Vet. Med. Sci. 55(4):549-54 (1993), which is herebyincorporated by reference in its entirety), and Aedes aegypti mosquitoesresistant to luciferase expression (Johnson et al., “Inhibition ofLuciferase Expression in Transgenic Aedes Aegypti Mosquitoes by SindbisVirus Expression of Antisense Luciferase RNA,” Proc. Natl. Acad. Sci.USA 96(23): 13399-403 (1999), which is hereby incorporated by referencein its entirety). Generally, pathogen resistance was mediated throughproduction of viral coat protein RNA in the above listed studies. Viralcoat protein genes, and fragments thereof, have been used successfullyin plants for RNA-mediated pathogen-derived resistance since presumably,the transcript is highly expressed and is very stable (Pang et al.,“Nontarget DNA Sequences Reduce the Transgene Length Necessary forRNA-Mediated Tospovirus Resistance in Transgenic Plants,” Proc. Natl.Acad. Sci. USA 94:8261-8266 (1997), which is hereby incorporated byreference in its entirety). It was demonstrated that only a portion ofthe coat protein gene was required to confer resistance against theviral pathogen. For example, a minimum length (somewhere between 236-387bp) of the gene for the 29 Kd nucleocapsid protein of tomato spottedwilt virus (TSWV) was required to develop RNA-mediated resistance intransgenic Nicotiana benthamiana plants (Pang et al., “Nontarget DNASequences Reduce the Transgene Length Necessary for RNA-MediatedTospovirus Resistance in Transgenic Plants,” Proc. Natl. Acad. Sci. USA94:8261-8266 (1997), which is hereby incorporated by reference in itsentirety). It was also determined that any region of the coding sequencefor the TSWV nucleocapsid protein can be used to develop virusresistance (Pang et al., “Nontarget DNA Sequences Reduce the TransgeneLength Necessary for RNA-Mediated Tospovirus Resistance in TransgenicPlants,” Proc. Natl. Acad. Sci. USA 94:8261-8266 (1997), which is herebyincorporated by reference in its entirety).

Animals suitable for this aspect of the present invention include,without limitation, those selected from the group consisting of marinefish; crustaceans, including prawns and shrimp; shellfish; and insects.

The present invention also relates to a method of regulating the growthof an animal. This involves transforming an animal with a nucleic acidconstruct of the present invention having the actin or β-actin promoterof the present invention operably linked to a nucleic acid moleculeencoding a growth regulating protein, and a 3′ regulatory region.Nucleic acid molecules suitable for this aspect of the present inventioninclude those that encode proteins that up-regulate growth anddown-regulate growth. Examples of suitable proteins that can be used toup-regulate growth include growth hormones, including withoutlimitation, the androgenic hormone. Animals suitable for this aspect ofthe present invention include, without limitation, those selected fromthe group consisting of marine fish; crustaceans, including prawns andshrimp; shellfish; and insects.

Another aspect of the present invention is a method of increasing stresstolerance in an animal, including stress induced by cold. This involvestransforming an animal with the nucleic acid construct of the presentinvention having the actin or β-actin promoter of the present inventionoperably linked to a nucleic acid molecule encoding protein and a 3′regulatory region. Nucleic acid molecules suitable for this aspect ofthe present invention include those encoding for a protein thatincreases stress tolerance in an animal. An exemplary protein would be aheat shock protein, such as HSP70 or HSP26, which may enhance coldtolerance in an animal. Animals suitable for this aspect of the presentinvention include without limitation, those selected from the groupconsisting of marine fish; crustaceans, including prawns and shrimp;shellfish; and insects.

The present invention also relates to a nucleic acid construct havingthe isolated nucleic acid molecule encoding β-actin from shrimp having anucleotide sequence of SEQ ID NO: 2, and an expression vector and hostcells transduced with such a nucleic acid construct. In this aspect ofthe present invention, preparation of nucleic acid construct, vector,and host cells is carried out as described above for nucleic acidconstructs, vector, and host cells in earlier aspects of the presentinvention, including the choice of suitable vectors, 3′ regulatoryregions, other regulatory element(s) when appropriate, and host cells,or in accordance with molecular biology methods available in the art,with the exception of the nucleic acid promoter molecule. The nucleicacid promoter molecule used in the nucleic acid construct of this aspectof the present invention may be one of the promoter molecules of thepresent invention, for example, the actin or β-actin nucleic acidpromoters of the present invention. Other promoters are also suitable,including those that are constitutive, inducible or repressible.Examples of some constitutive promoters that are widely used forinducing expression of transgenes include the nopoline synthase (“NOS”)gene promoter, from Agrobacterium tumefaciens, (U.S. Pat. No. 5,034,322to Rogers et al., which is hereby incorporated by reference in itsentirety), the cauliflower mosaic virus (“CaMV”) 35S and 19S promoters(U.S. Pat. No. 5,352,605 to Fraley et al., which is hereby incorporatedby reference in its entirety), the enhanced CaMV35S promoter (“enhCaMV35S”). Also suitable are those derived from any of the severalpreviously identified actin genes, which are known to be expressed inmost cells types (U.S. Pat. No. 6,002,068 to Privalle et al., which ishereby incorporated by reference in its entirety), and the ubiquitinpromoter (“ubi”), which is a gene product known to accumulate in manycell types. Promoters for this aspect of the present invention arechosen with regard to the desired application of the nucleic acidconstruct, and are incorporated into the nucleic acid construct asdescribed above or by using standard cloning procedures known in theart, such as described by Sambrook et al., Molecular Cloning: ALaboratory Manual, Third Edition, Cold Spring Harbor: Cold Spring HarborLaboratory Press, New York (2001), and U.S. Pat. No. 4,237,224 to Cohenand Boyer, which are hereby incorporated by reference in their entirety.

Another aspect of the present invention relates to a nucleic acidexpression cassette containing an isolated actin nucleic acid promotermolecule of the present invention, a multiple cloning site, an operabletermination segment, and a nucleic acid molecule encoding a detectablemarker. In this aspect, a nucleic acid expression cassette is preparedgenerally as described for the making of the nucleic acid constructhaving the actin promoter of the present invention, with the promotermolecule and a suitable 3′ termination segment (meaning apolyadenylation signal and a termination signal); however, the promoteris incorporated into a vector having a multiple cloning site (MCS) forthe insertion of one or more nucleic acid molecules of choice by a user.In one embodiment, the expression cassette also contains a detectablemarker. Exemplary markers include, without limitation, green fluorescentprotein, enhanced green fluorescent protein, β-galactosidase, andluciferase. The promoter molecule, 3′ termination segment, anddetectable marker, if desired, are ligated into a vector having a MCS,using standard cloning procedures known in the art, such as described bySambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition,Cold Spring Harbor: Cold Spring Harbor Laboratory Press, New York(2001), and U.S. Pat. No. 4,237,224 to Cohen and Boyer, which are herebyincorporated by reference in their entirety.

EXAMPLES Example 1 Animals

Live shrimp and fertilized eggs of L. vannamei were obtained from alocal aquafarm in Hawaii. Immediately after fertilization, the shrimpeggs were collected with a fine mesh net and concentrated by a briefcentrifugation at 1000 g for 20 seconds, then transferred to 1.5 mlsterilized sea water in a small dish and subjected to micro-injection.

Example 2 Micro-Injection Procedures

Procedures of micro-injection followed the methods of Chong et al.,“Expression and Fate of CAT Reporter Gene Microinjected into FertilizedMedaka (Oryzias latipes) Eggs in the Form of Plasmid DNA, RecombinantPhage Particles and its DNA,” Theor. Appl. Genet. 78:369-380 (1989),Penman et al., “Factors Effecting Survival and Integration FollowingMicro-Injection of Novel DNA in Rainbow Trout Eggs,” Aquaculture85:35-50 (1990); Collas et al., “Transferring Foreign Genes intoZebrafish Eggs By Microinjection,” In Houdebine, L. M. (ed.) TransgenicAnimals—Generation and Use Harwood Academic Publishers (In Press); whichare hereby incorporated by reference in their entirety), withmodifications. Briefly, microinjection was performed with the Femtojetmicroinjection system (Brinkmann Instruments, Inc., Westbury, N.Y.).Femtotip injection needles (Brinkmann Instruments, Inc.) were secured tothe micromanipulator (Drummond Scientific Co., Philadelphia, Pa.) madefrom borosilicate glass capillary tubes using a horizontal Sutter P-87puller. The needles were generally about 5 cm long, steep near theshoulder, and shallow close to the tip which is of 10-15 μm in diameter.Mineral oil was introduced into the base of the injection needle priorto being secured to the micro-manipulator (Drummond Scientific Co.,Philadelphia, Pa.). DNA solution (1 μg/μl in sterilized H₂O) wasintroduced into the needle with a microloader tip (BrinkmannInstruments, Inc.) through the tip by capillary action. Prior toinjection, the shrimp eggs were placed in the petri dish equipped with afine mesh grid, which serves as a barrier so that the eggs will not rollduring injection. The petri dish was then placed under a high powerstereo dissecting microscope (Carl Zeiss, Jena, Germany) with theinjection needle positioned at 45° above the horizontal axis. Eggs wereinjected with approximately 50 picoliters of DNA solution. Each injectordispenser released 4 ηl (working range is 4 ηl -40 η1) of DNA solutioninto the egg. With suitable adjustment of the micro-manipulator, asuitable rate of injection of about 5 injections per minute wasachieved. Several hundred eggs can be injected per hour with a singleneedle filling. A series of micro-injection experiments were performedfor testing transgene expression efficiency of several constructs whichcontain various regulatory regions of the shrimp β-actin5C gene.

Example 3 Sample Collection

Immediately following injection, the putative transformed eggs wereplaced in a one-liter container with aerated seawater at roomtemperature where hatching takes place in about one day. After hatching,larvae were transferred to a 5-gallon glass aquarium (16″L×8″W×10″H)with aerated seawater containing 0.15 ppm each of penicillin andstreptomycin. Control groups of shrimp eggs were treated identicallyexcept for injection with water alone. The techniques for raisingpenaeid shrimp from the egg to post-larvae generally followed themethods described by Mock et al., “Techniques for Raising Penaeid Shrimpfrom the Egg to Postlarvae,” Maricult. Proc. World Soc. 3:143-156(1972), Brown et al., “The Maturation and Spawning of Penaeusstylirostris Under Controlled Laboratory Conditions,” Proc. WorldMaricult. Soc. 11:488-499 (1980), and Wyban et al., “Intensive ShrimpGrowth Trials in a Round Pond,” Aquaculture 76:215-225 (1989) (which arehereby incorporated by reference in their entirety). The shrimp were fedwith algae feed, and observed closely throughout the experimentalperiod. Animals were examined or sacrificed at several stages duringgrowth and development for analysis of transgene expression.

Example 4 Reverse Transcription-Polymerase Chain Reaction

Live shrimp (L. vannamei) were obtained from a local aquafarm in Hawaii.Approximately 2 μg of total RNA from shrimp tissue was used for reversetranscription reaction, and RT-PCR assays were carried out according tothe protocol of the GeneAmp RNA PCR Kit (Perkin-Elmer Cetus, Norwalk,Conn.), with slight modifications as described previously (Sun,“Molecular Cloning and Sequence Analysis of a cDNA Encoding aMolt-Inhibiting Hormone-like Neuropeptide from the White Shrimp Penaeusvannamei,” Mol. Mar. Biol. Biotechnol. 3(l):1-6 (1994), which is herebyincorporated by reference in its entirety). Amplifications wereperformed by a DNA Thermal Cycler (Perkin-Elmer 9600) programmed atsuitable temperatures for annealing and extension. A pair of degenerateprimers (P1 and P2) were constructed based on unique sequences tocytoplasmic actin5C protein of D. melanogaster (Fyrberg et al., “TheActin Genes of Drosophila: Protein Coding Regions are Highly Conservedbut Intron Positions Are Not,” Cell 24: 107-116 (1981); Bond et al.,“The Drosophila Melanogaster Actin 5C Gene Uses Two TranscriptionInitiation Sites and Three Polyadenylation Sites to Express MultiplemRNA Species,” Mol. Cell Biol. 6(6):2080-2088 (1986), which are herebyincorporated by reference in their entirety) and were used for PCRamplification. The oligonucleotide sequences of P1 (1735-1762) and P2(1959-1933) are as follows: (SEQ ID NO: 7) sense; P1:5′CTTACAAAATGTGT(C)GAC(T)GAA(G)GAA(G)GTIGC 3′ (SEQ ID NO: 8) antisenseP2: 5′CCG(A)TGC(T)TCG(AT)ATIGGG(A)TAC(T)TTIAGIGT 3′

Example 5 Southern Hybridization

The PCR-amplified DNA products (10 μl) were separated by electrophoresisin a 2% low melting temperature agarose gel containing ethidium bromide(0.5 μg/ml). After electrophoresis, the DNA was transferred to Hybond-N⁺membrane (Amersham, Piscataway, N.J.). Hybridization was performed at42° C. for 18 hours with α³²P-labeled actin5C-cDNA probe (actin5C-cDNAfrom Drosophila) in a solution containing 50% formamide, 6×SSPE(1×SSPE=0.15 MNaCl, 10 mMNaH₂PO₄, 1 mM ethylenediaminetetraacetic acid,pH 7.4), 5× Denhardt's reagent, 0.5% sodium dodecyl sulfate (SDS), and100 μg/100 ml denatured salmon sperm DNA with an actin5C-cDNA labeled byrandom priming with α³²P-dCTP as probe. After hybridization, the filterwas washed two times for 15 minutes in 2×S SSPE and 0.2% SDS at 42° C.,then two times for 15 minutes in 0.1×SSPE and 0.1% SDS at 68° C., andexposed to Kodak XAR-5 film at −80° C. for 10 hours.

Example 6 Cloning and Sequencing of DNA

The target DNA fragment, as identified by Southern hybridization, wascloned with the TA cloning kit (Invitrogen, Carlsbad, Calif.). Briefly,the PCR-product was ligated into the TA cloning vector, pCRII. One Shotcompetent cells were used for transformation. Positive white colonieswere picked and analyzed by miniprep to verify the presence of clonedPCR product. Standard protocols for ligation, cloning, andtransformation followed Sambrook et al., Molecular Cloning, A LaboratoryManual, Second edition, New York: Cold Spring Harbor Laboratory Press(1989). After purification using a QIAprep Spin Plasmid Miniprep Kit(Qiagen, Valencia, Calif.), DNA was sequenced by thedideoxy-chain-termination method (Sanger et al., “DNA Sequencing withChain-Terminating Inhibitors,” Proc. Natl. Acad. Sci. USA 74:5463-5467(1977), which is hereby incorporated by reference in its entirety) usingthe Sequenase system (version 2.0, USB, West Conshocken, Pa.) and/or theautomatic sequencing method using the DyeDeoxy Terminator cyclesequencing kit (Applied Biosystems, Foster City, Calif., model 373A).Each DNA sample was sequenced twice in each direction for sequenceconfirmation.

Example 7 Library Screening

The existing shrimp genomic library, constructed using the LambdaGEM-11vector and containing 360,000 recombinant clones, was first used forscreening the genomic clone of actin5C. The relevant facts are that theactin5C gene is abundant in cytoplasm. Therefore, it was probable thatthe gene was present in the partial genomic library of 360,000recombinant clones, and the vector is LambdaGEM-11 which contains thelengths of inserts between 9-23 kb. The known actin5C of Drosophila is17.5 kb.

The genomic library was screened using a combination of PCRamplification (Amaravadi et al., “A Rapid and Efficient, NonradioactiveMethod for Screening Recombinant DNA Libraries,” Biotechniques16(l):98-103 (1994), which is hereby incorporated by reference in itsentirety) and the in situ plaque hybridization technique (Benton et al.,“Screening Lambda gt Recombinant Clones by Hybridization to SinglePlaques in situ,” Science 196(4286):180-182 (1977), which is herebyincorporated by reference in its entirety) using primer 1 and primer 2(described in Example 4) and the RT-PCR generated DNA fragment as aprobe. Briefly, about 1×10⁶ recombinant clones were plated on 20 (150mm) plates and incubated for 7-10 hours at 37° C. or until plaques beginto contact each other. The phages were soaked in 10 ml of phage dilutedbuffer (PDB) overnight at 4° C., the PDB collected from each plate, andcentrifuged at 5,000×g for 10 min to remove debris. E. coli were lysedby adding a few drops of CHCl₃. An aliquot (1 μl) of plate lysate wasused as the template for PCR assay. The PCR protocol was performed aspreviously described (Sun, “Molecular Cloning and Sequence Analysis of acDNA Encoding a Molt-Inhibiting Hormone-like Neuropeptide from the WhiteShrimp Penaeus vannamei,” Mol. Mar. Biol. Biotechnol. 3(1):1-6 (1994),which is hereby incorporated by reference in its entirety) and the PCRproducts were first analyzed by agarose gel electrophoresis. Thedetection of an expected 224-bp DNA product indicated a positive actin5Cclone in the plate lysate. Once a positive plate lysate was identified,several rounds of replating and PCR amplification led to theidentification of individual positive plaques. Individual positiveplaques were confirmed by plaque hybridization as described by Sambrooket al., Molecular Cloning. A Laboratory Manual, 2^(nd) ed., Cold SpringHarbor, N.Y.: Cold Spring Harbor Laboratory Press (1989), which ishereby incorporated by reference in its entirety, using thePCR-generated DNA labeled with α³²P-dCTP as a probe. The sensitivity andreliability of plaque hybridization demonstrated that a positive actin5Cclone was obtained.

Phagemid DNA from positive clones were isolated using Wizard Lamb &Preps DNA Purification system (Promega, Madison, Wis.), and subjected toSouthern analysis. Positive clones having the largest size of DNA wereselected and their DNAs multiplied in E. coli, purified, and the DNAswere further characterized by sequencing and physical mapping. Thenucleotide sequence of the shrimp actin5C gene was also analyzed fortranscription factor binding motifs using the Find patterns and forsequence comparison with the chicken β-actin promoter and fly β-actinpromoters using the Best-Fit program (Genetics Computer Group, Madison,Wis.).

For information purposes, the shrimp cDNA library was also screened forcDNA(s) encoding the actin5C protein using the same probe andstrategies. The shrimp actin5C-cDNA(s) isolated from positive clones waspurified and sequenced and their deduced amino acid sequences analyzedand compared with published data from other species.

Example 8 Mapping of the Transcriptional Start Site by Primer Extension

To determine the transcription start site, primer extension wasperformed using a AMV-reverse transcriptase primer extension system(Promega, Madison, Wis.). A 5′-end-labeled antisense oligonucleotidecomplementary to the part of the 5′-flanking region of the shrimpactin5C gene was incubated with 30 μg of total RNA isolated from shrimpembryos for 24 hours. After annealing at 62° C. for 20 minutes,AMV-reverse transcriptase extension mix was added to the annealedprimer/RNA followed by a 30 minute incubation at 42° C. The resultingcDNA was analyzed by electrophoresis on a 8% sequencing gel and the sizeof the primer extended product determined by an end-labeled φ×174 Hinf 1DNA-marker.

Example 9 Northern Hybridization

In order to verify that the shrimp act5C gene obtained was cytoplasmic,Northern hybridization experiments were performed to study the temporaland spatial expressions of the shrimp act5C gene during the shrimp lifecycle. Poly(A)⁺RNA was prepared from several developmental stages,ranging from early embryo to adult (i.e. embryos, larvae, pupae,juvenile, and adult) and from various organs and tissues (i.e. brain,eyestalk, stomach, heart, hepatopancreas, ovary, and leg muscle). Equalamounts of poly(A)⁺RNA from different developmental stages and fromvarious organs were subjected to gel electrophoresis under denaturingconditions, transferred to nitrocellulose filters, and hybridized to³²P-labeled shrimp act5C-cDNA under conditions that are sufficientlystringent for specificity. Similar procedures of Northern hybridizationas described in Sun, “Molecular Cloning and Sequence Analysis of a cDNAEncoding a Molt-Inhibiting Hormone-like Neuropeptide from the WhiteShrimp Penaeus vannamei,” Mol. Mar. Biol. Biotechnol. 3(1):1-6 (1994),which is hereby incorporated by reference in its entirety, were used.

Total RNA from each shrimp sample was isolated according to the methodof Chomczynski et al., “Single-Step Method of RNA Isolation by AcidGuanidinium Thiocyanate-Phenol-Chloroform Extraction,” Anal. Biochem.162(1):156-159 (1987), which is hereby incorporated by reference in itsentirety, and Poly(A⁺) RNA will be obtained using the Poly(A) Quik mRNAPurification kit (Stratagene, LA Jolla, Calif.) andspectrophotometrically quantitated. RNAs to be separated were denaturedby heating for 15 minutes at 65° C. One μg/lane was loaded on a 1.2%agarose-0.66 M formaldehyde gel (Lehrach et al., “RNA Molecular WeightDeterminations by Electrophoresis Under Denaturing Conditions, aCritical Reexamination,” Biochemistry 16:4743-4751 (1977). Theelectrophoresis buffer consisted of 20 mM Na-MOPS (Sigma, St. Lous,Mo.), 5 mM NaOAc, 1 mM EDTA. After electrophoresis, the gel was blottedto a nylon membrane (Amersham, Piscataway, N.J.) in 10×SSPE. Afterblotting for 20 hours, filters were air dried, then baked for 2 hours ina vacuum oven.

Filters were pre-hybridized at 50° C. for 4 hours in a solutioncontaining 50% (v/v) deionized formamide, 6×SSPE, 5× Denhardt's reagent,0.5% SDS, and 100 μg/ml denatured salmon sperm DNA, then hybridized tothe random primed labeled ³²P-act5C-cDNA in the buffer above at 50° C.for 20 hours. After hybridization, filters were washed twice at roomtemperature in 2×SSPE, 0.5% SDS, twice at 75° C. in 0.2×SSPE, 0.05% SDS,and exposed to Kodak XAR-5 X-ray film plus intensifying screens at −80°C.

Example 10 Transient Gene Expression

Transient gene expression of the EGFP gene in transgenic shrimp wasmonitored by fluorescent microscope examination. Due to the spectralproperties of EGFP which absorbs blue light and emits green light, theexpression of the EGFP can be visualized by placing the live shrimp on adark disk under a fluorescence microscope (Leitz) adapted with a filterset (excitation wavelength of 490 nm and emission wavelength of 525 nm).The intensity of the fluorescence correlated to the EGFP level can bedocumented by photography.

The survival rate and the number of fluorescent eggs were determined,and the results from different promoter-regions constructs, fromdifferent animal handling conditions, and from the controls werecompared.

Example 11 Measurement of Endogenous Fluorescence in Shrimp viaFluorescence Microscopy

Fluorescence microscopy images using fluorescein and rhodamine filtersets illustrated the relatively high levels of visible, endogenousfluorescence in the hepatopancreas and proximal regions of the animals.Furthermore, the endogenous fluorescence appears to increase as theanimal matures. In order to determine whether EGFP fluorescence could bedetected against the background of endogenous fluorescence in theshrimp, spectrofluorometric measurements were taken. Fluorescencemicroscopy was performed on live, whole shrimp at each of fourdevelopmental stages (egg, protozoea, mysis, and postlarvae) using afluorescence inverted microscope (Zeiss Axiovert 10). Images of eachshrimp viewed under brightfield, fluorescein filter set (excitation:320-500 nm, emission: 505-560 nm) and rhodamine filter set (excitation:370-590 nm, emission: 570-600 nm), were captured with a 12-bit CCDcamera system (Photometrics). These shrimp were also photographed with adigital camera (Nikon) mounted to a stereo dissection microscope (Zeiss,Jena, Germany).

Example 12 Monitoring EGFP and DsRed Reporter Gene Expression viaSpectrofluorometry

Plasmid DNA consisting of 4.5 μg of the vector EGFP-N1, in 2 μl 10 mMTris, pH 7.8, was injected into juvenile shrimp (4 cm in length) at thesecond abdominal muscle segment under the exoskeleton. This procedurewas repeated using the vector, 2-N1 (Clontech Laboratories, Inc., PaloAlto, Calif.). The DsRed vector encodes the red fluorescent protein fromDiscosoma sp. Two days after injection, injected tissue segments wereexcised from shrimp and homogenized. Fluorescent intensity of thehomogenate supernatant was measured using a fluorescencespectrophotometer (F-2500, Hitachi) at appropriate wavelengths(excitation: 488 nm, emission: 507 nm for EGFP; and excitation: 558 nm,emission: 583 nm for DsRed). Results from the analysis of EGFP and DsRedexpression efficiency in shrimp via muscular injection, is shown inFIGS. 3A-B. Expression of both EGFP and DsRed are approximately 2 timeshigher than fluorescence of the controls, demonstrating theirsuitability as a marker gene. However, EGFP may be preferable to DsRedas a marker gene since greater variability and inaccuracy may beassociated with DsRed's low fluorescent intensity values and theextended protein maturation time (˜20 hrs).

Example 13 Gene Integration and Expression

The expression of the GFP gene in the egg, larva, and juvenile werefollowed by fluorescent microscopy as described above, and also byspectrofluorescent measurement. The GFP in the protein extract wasquantified by measuring emission at 509 nm when exited at 395 nm using aspectrofluorometer (Kratos FS 970). Fluorescence intensity wasnormalized to protein concentration as determined by Bradford assayusing the Bio-Rad protein assay kit (Bio-Rad Lab, Hercules, Calif.). ForSouthern hybridization, genomic DNA was isolated from the control andputative transformed shrimp using Easy DNA kit (Invitrogen, Carlsbad,Calif.). After digestion with appropriate restriction enzyme(s), the DNAwas subjected to gel electrophoresis, transferred to a Nylon membrane,and fixed with UV light cross-linking. Blots were hybridized with theGFP DNA fragment labeled with Digoxigenin as described by Sun,“Expression of the Molt-Inhibiting Hormone-like Gene in the Eyestalk andBrain of the White Shrimp Penaeus vannamei,” Mol. Mar. Biol. Biotechnol.4(3):262-268 (1995), which is hereby incorporated by reference in itsentirety. Genomic DNAs isolated from the transgenic animals were used astemplates for polymerase chain reaction assay (Sun, “RecombinantMolt-Inhibiting Hormone-like Neuropeptide Produced in the Yeast Pichiapastoris,” In: PACON International Proceedings. Aug. 5-8, 1997, HongKong, pp. 509-518 (1997), which is hereby incorporated by reference inits entirety) to confirm the GFP gene has integrated into the shrimpgenome. To detect and localize the GFP transcripts in the transgenicshrimp, techniques of SDS-polyacrylamide gel electrophoresis, Westernblot analysis and in situ hybridization of the shrimp tissue sectionswere performed Sun, “Expression of the Molt-Inhibiting Hormone-like Genein the Eyestalk and Brain of the White Shrimp Penaeus vannamei,” Mol.Mar. Biol. Biotechnol. 4(3):262-268 (1995), Sun, “RecombinantMolt-Inhibiting Hormone-like Neuropeptide Produced in the Yeast Pichiapastoris,” In: PACON International Proceedings. Aug. 5-8, 1997, HongKong, pp. 509-518 (1997), which are hereby incorporated by reference intheir entirety).

Example 14 Identification and Characterization of a Gene-Specific DNAFragment Encoding the Cytoplasmic β-actin from Shrimp, L. vannamei

Actin is a major protein constituent of all eukaryotic cells. Invertebrates at least six actin variants have been characterized: twofrom smooth muscles, two from striated muscles, and two from non-muscletissues (β and γ) (Vandekerckhove et al., “The Complete Amino AcidSequence of Actins from Bovine Aorta, Bovine Heart, Bovine Fast SkeletalMuscle, and Rabbit Slow Skeletal Muscle. A Protein-Chemical Analysis ofMuscleActin Differentiation,” Differentiation 14(3):123-133 (1979),which are hereby incorporated by reference in their entirety). Althoughthe actin gene family is expressed in all tissues, individual actingenes show tissue and developmental specificity in their expression(Fyrberg et al., “Transcripts of the Six Drosophila Actin GenesAccumulate in a Stage-and Tissue-Specific Manner,” Cell 33(1):115-123(1983); Sanchez et al., “Two Drosophila actin Genes in Detail: GeneStructure, Protein Structure, and Transcription During Development,” J.Mol. Biol. 163:533-551 (1983); Vandekerckhove et al., “Chordate MuscleActins Differ Distinctly from Invertebrate Muscle Actins. The Evolutionof the Different Vertebrate Muscle Actins,” J. Mol. Biol. 179(3):391-413(1984), which are hereby incorporated by reference in their entirety).There are also six actin genes found in the invertebrate fly, Drosophilamelanogaster. Two of the Drosophila actin genes, act5C and act42A, areexpressed in undifferentiated cells and encode cytoplasmic or non-muscleactins (Fyrberg et al., “Transcripts of the Six Drosophila Actin GenesAccumulate in a Stage-and Tissue-Specific Manner,” Cell 33(1):115-123(1983), which is hereby incorporated by reference in its entirety). Theremaining four genes probably respond to regulatory molecules and aresynthesized during early muscle cell differentiation. These invertebratecytoplasmic actin genes are different from vertebrate non-muscle actingenes in terms of amino acid sequences and isoelectric points of theprotein molecules. However, β-actin is the major non-muscle orcytoplasmic actin isoform and it is expressed in most eukaryoticnon-muscle cells, as well as in undifferentiated myoblasts. And, becauseβ-actin promoter is an active cellular promoter (Gunning et al., “AHuman β-Actin Expression Vector System Directs High-Level Accumulationof Antisense Transcripts,” Proc. Natl. Acad. Sci. USA 84:4831-4835(1987), which is hereby incorporated by reference in its entirety) andhas constitutive expression properties, β-actin gene(s) are a primetarget for transgenic manipulation technology.

Reverse-transcriptase-polymerase chain reaction (RT-PCR) method (Sun,“Expression of the Molt-Inhibiting Hormone-like Gene in the Eyestalk andBrain of the White Shrimp Penaeus vannamei,” Mol. Mar. Biol. Biotechnol.4(3):262-268 (1995)) was used to generate a DNA fragment encoding thepartial β-actin5C from shrimp tissue, as described in Example 4.Degenerate primer pairs P1-P2, described in Example 4, were constructedagainst conserved regions of the fruit fly, Drosophila melanogasteractin5C protein (Bond et al., “The Drosophila Melanogaster Actin 5C GeneUses Two Transcription Initiation Sites and Three Polyadenylation Sitesto Express Multiple mRNA Species,” Mol. Cell Biol. 6(6):2080-2088(1986), which are hereby incorporated by reference in their entirety).Total RNA isolated from shrimp tissue using the method described byChomczynski et al., “Single-Step Method of RNA Isolation by AcidGuanidinium Thiocyanate-Phenol-Chloroform Extraction,” Anal. Biochem.162(1):156-159 (1987), which is hereby incorporated by reference in itsentirety, was used as template for the RT-PCR reaction. Size analysis ofRT-PCR products by ethidium bromide-agarose gel electrophoresis revealeda high intensity DNA band of about 224 bp, which was the size expectedusing the P1/P2 primer set. The PCR-generated DNA product was purifiedfrom the agarose gel, cloned with a pCR2.1 vector using the original TAcloning kit (Invitrogen, Carlsbad, Calif.). The PCR-generated 224-bp DNAfragment encoding the shrimp β-actin5C was amplified, purified andlabeled using the methods described by Sun (Sun, “Molecular Cloning andSequence Analysis of a cDNA Encoding a Molt-Inhibiting Hormone-likeNeuropeptide from the White Shrimp Penaeus vannamei,” Mol. Mar. Biol.Biotechnol. 3(1):1-6 (1994), Sun, “Expression of the Molt-InhibitingHormone-like Gene in the Eyestalk and Brain of the White Shrimp Penaeusvannamei,” Mol. Mar. Biol. Biotechnol. 4(3):262-268 (1995), which arehereby incorporated by reference in their entirety), and used as aneffective probe to identify the full-length cDNA and the genomic clonesof the shrimp β-actin5C gene by screening the existing cDNA and genomiclibraries of the shrimp L. vannamei. The full-length genomic DNAsequence of the shrimp β-actin gene was generated via PCR using primerswhose sequences were based on a region of the promoter and the 3′untranslated region of the shrimp β-actin cDNA. The PCR productgenerated was then used as a template in a 2^(nd) round of the PCR usingnested primers whose sequences were based on the shrimp β-actin cDNA.

Example 15 Species Comparison of the Partial Amino Acid Sequence ofShrimp β-actin5C

In comparing the partial deduced amino acid sequence of the shrimpβ-actin5C with other cytoplasmic β-actin proteins from other species, itwas found that the shrimp β-actin5C shares more than 90% homology inamino acid sequences studied with crab (C. carnifex), fly (D.Melanogaster), nematode (C. elegans), and chicken. It was also notedthat a set of eleven amino acid residues in the shrimp β-actin5C isfound missing at position #24 to position #34; and an additional aminoacid, aspartic acid, is present at position #59.

Example 16 Spatial Expression of the Partial Shrimp β-actin5C DNA

Transcripts from the partial β-actin5C gene are found in most of theshrimp system including eye, stomach, heart, and hepatopancreas whenusing the RT-PCR technique for the detection. Expression of the shrimpβ-actin5C gene is especially abundant in hepatopancreas but noexpression was found in muscle. This observation suggests that theshrimp β-actin5C transcript is present in organs of non-muscle type andis thought to be a cytoplasmic form of actin.

Example 17 Construction and Screening of the Shrimp Genomic Library

A genomic library of the Pacific white shrimp L. vannamei (1.2×10⁶recombinants) was constructed with the LambdaGEM-11 vector (Promega,Madison, Wis.) using genomic DNA prepared by Easy DNA kit (Invitrogen,Carlsbad, Calif.). The purified genomic DNA was partially digested bySau3A and fragments of 15-23 kb were ligated into the LambdaGEM-11vector. Packaging was performed using the Packagene Extract system(Promega, Madison, Wis.).

Approximately 1×10⁴ plaques were screened by a combination of PCRamplification method (Amaravadi et al., “A Rapid and Efficient,Nonradioactive Method for Screening Recombinant DNA Libraries,”Biotechniques 16(1):98-103 (1994), which is hereby incorporated byreference in its entirety) and the in situ plaque hybridizationtechnique (Benton et al., “Screening Lambda gt Recombinant Clones byHybridization to Single Plaques in situ,” Science 196(4286):180-182(1977), which is hereby incorporated by reference in its entirety) fromthe shrimp genomic library. Primers were made based on the 224-bp DNAsequence (see Example 14) for the PCR assay. The PCR-generated 224-bpDNA fragment was used as a probe for in situ plaque hybridization. Atotal of twelve positive clones were isolated. The positive genomicclones were grown and the bacteriophage DNAs were prepared by usingλ-DNA purification kit (Stratagene, La Jolla, Calif.). Purified phageDNA were analyzed on Southern blot. The sizes of phage DNA as revealedby ethidium bromide staining and UV illumination after agarose gelelectrophoresis was ranged from 1.0 to 18 kd. The positive restrictionenzymes digested fragments were selected and subcloned into theBluescript vector (Stratagene, La Jolla, Calif.) for DNA sequencing andanalysis. These DNA samples were then processed for DNA sequencing andassembling.

Example 18 β-Actin Promoter Sequence Identification

Using the gene walking method, a 1297-bp promoter of the shrimp β-actingene was identified and sequenced. This promoter contains a CAAT box,TATA box, and CArG sequence that are characteristic of β-actin promotersfound in other organisms. This promoter, termed β-ActinP2, identifiedherein as having SEQ ID NO: 1, was cloned and used in vectorconstruction. A full-length cDNA encoding the β-actin (Genbank AccessionNo. AF300705) and its promoter sequence from the Pacific white shrimp L.vannantei was also identified, cloned, and sequenced (Genbank AccessionNo. AF300705). The cDNA for β-Actin is identified herein as SEQ ID NO:2.

Example 19 Actin Promoter Sequence Identification

The existing shrimp genomic library was screened for the genomic cloneof actin using a combination of PCR amplification method (Amaravadi etal., “A Rapid and Efficient Nonradioactive Method for ScreeningRecombinant DNA Libraries,” Biotechniques 16(1):98-103 (1994), which ishereby incorporated by reference in its entirety) and the in situ plaquehybridization technique (Benton et al., “Screening Lambdagt RecombinantClones by Hybridization to Single Plaques in situ,” Science196(4286):180-182 (1977), which is hereby incorporated by reference inits entirety). The PCR-generated 224-bp DNA fragment (See Example 14)labeled with digoxigenin was used as a probe for non-radioactive in situplaque hybridization. The positive genomic clones were isolated, grown,and the bacteriophage DNAs were prepared using λ-DNA purification kit(Qiagen, Inc., Valencia, Calif.). The positive restriction enzymedigested fragments were selected and subcloned into the Bluescriptvector (Stratagene, La Jolla, Calif.) for DNA sequencing and analysis.These DNA samples were then processed for DNA sequencing and assembling.

The shrimp actin promoter (SEQ ID NO: 4) contains TATA and CAAT boxesapproximately 500 base pairs upstream from the translation start site.Unique CACA-rich and CATA-rich regions are located in the actin promoterregion upstream from the expected TATA and CAAT boxes. The deducedpolypeptide of the shrimp actin consists of a 64-amino acid signalpeptide and a 311-amino acid mature polypeptide. This shrimp actinexhibits 94% amino acid homology with the tiger prawn (Penaeus monodon)actin, 93% homology with the rattail fish (Coryphaenoides acrolepis)skeletal alpha actin type 2, and 93% homology with human (Homo sapiens)alpha actin of the cardiac muscle.

Example 20 Actin cDNA Sequence Identification

The deduced polypeptide of the shrimp actin consists of a 64-amino acidsignal peptide and a 311-amino acid mature polypeptide. This shrimpactin exhibits 94% amino acid homology with the tiger prawn (Penaeusmonodon) actin, 93% homology with the rattail fish (Coryphaenoidesacrolepis) skeletal alpha actin type 2, and 93% homology with human(Homo sapiens) alpha actin of the cardiac muscle.

Example 21 Expression of a Reporter Gene in Shrimp Muscle by Injection

In order to test the ability of a heterologous promoter to driveexpression of a reporter gene and to investigate parameters ofintroducing exogenous DNA into shrimp system, a trial experiment wasperformed in which an expression vector containing a promoter of humancytomegalovirus (CMV) sequence and a reporter gene of β-galactosidase(β-Gal) was prepared and delivered into shrimp muscle via directinjection. Injection was performed with a 33-gauge hypodermic needlefilled with various amount of super-coiled plasmid DNA in 2.5 ulPantin's saline buffer (Pantin, 1934, which is hereby incorporated byreference in its entirety) into the fourth tail segment of juvenilewhite shrimp (approximately 5-6 inches in length). The expressionefficiency was monitored spectrophotometrically using theβ-Galactosidase enzyme assay system (Promega, Madison, Wis.). Musclebiopsy samples were taken for determining the level of expression at dayone through day ten after plasmid DNA injection. Control muscle samplesfrom shrimp injected with Pantin's saline buffer alone were also taken.Most of the samples from shrimp injected with the pCWV-β-Gal showedβ-Gal activity upon assay, whereas no significant activity was observedin control samples. The survival rate was found to be 95% in a total of76 animals tested. Expression of the reporter gene as monitoredspectrophotometrically was observed 24 hours after injection withhighest expression at day two. The exogenous DNA of β-galactosidase wasdetected by polymerase chain reaction four days after injection. Theseresults demonstrated that micro-injection into shrimp muscle is apotential technique for testing transient expression of foreign gene inshrimp system.

Example 22 Generating DNA Fragments Encoding the TSV-CP by RT-PCR

The genomic organization of TSV consists of a linear, positive-sense,single stranded RNA of approximately 9 kb in length. Its capsid consistsof three major polypeptides (24, 40, and 55 Kd) and one minorpolypeptide (58 Kd) (Mari et al., “Full Nucleotide Sequence and GenomeOrganization of the Taura Syndrome Virus of Penaeid Shrimp,” Unpublished(2000); Genbank Accession Number: AF277675, which are herebyincorporated by reference in their entirety). One of the genes encodedby the RNA is a 111 Kd viral coat protein (Genbank Accession #AF277378,which is hereby incorporated by reference in its entirety). This coatprotein is most likely cleaved co- and post-translationally since theproteinic capsid of purified TSV was found to consist of three major(55, 40, and 24 Kd) polypeptides and one minor (58 Kd) polypeptide(Bonami et al., “Taura Syndrome of Marine Penaeid Shrimp:Characterization of the Viral Agent,” J. Gen. Virol. 78:313-319 (1997),which is hereby incorporated by reference in its entirety). This geneencoding the structural coat protein was selected as a prime candidatefor developing of viral protection in shrimp. Total RNA was isolatedfrom TSV-infected shrimp. Several gene specific oligonucleotide primerswere synthesized based on the published TSV coat protein (TSV-CP) genesequence (Genbank Accession No. AF277378, which is hereby incorporatedby reference in its entirety). Use of these gene specific primers andTSV RNA in the RT-PCR assay yielded distinct, high-intensity bands,corresponding to the expected sizes, as shown in FIG. 4. These DNAfragments were purified, cloned, sequenced, and various fragments fromabout 500 to 1000 bp of the TSV coat protein gene in both positive andnegative orientations were used in vectors constructed for transfer intoshrimp.

Example 23 Generating DNA Fragments Encoding the IHHNV-CP by RT-PCR

IHHNV is a single-strand DNA virus with a viral coat protein of 37.5 Kd(Genbank Accession #AF218266, which is hereby incorporated by referencein its entirety). Eight oligonucleotides, gene specific to the IHHNV,were synthesized (Biotechnology/Molecular Biology InstrumentationFacility, University of Hawaii) based on the published nucleotidesequences of the IHHNV gene and were used as primers in the RT-PCRassays. Shrimp samples infected with IHHNV were obtained from DeeMontgomery-Brock (Aquaculture Development Program, Department ofAgriculture, State of Hawaii). Approximately 0.25 g of the muscle tissuewere ground into powder in liquid nitrogen and total RNA was isolatedusing the Purescript RNA isolation kit (Gentra Systems, Inc.), and usedas template in RT-PCR. The RT-PCR assays were performed according to theprocedures described by Sun (Sun, “Expression of the Molt-InhibitingHormone-like Gene in the Eyestalk and Brain of the White Shrimp Penaeusvannamei,” Mol. Mar. Biol. Biotechnol. 4(3):262-268 (1995), which ishereby incorporated by reference in its entirety) using the GeneAmp RNAPCR Kit (PE Biosystems, Foster City, Calif.). Several DNA bands weregenerated via RT-PCR assays using the synthesized primers and theIHHNV-RNA as template. Results are summarized in Table 1, below, andshown in FIG. 5. TABLE 1 IHHNV Gene Specific Oligonucleotide Primers SEQDNA size ID Gener- Primer NO: Sequence Expected ated 1 9 5′-CAA ACT ATGAAG ACC CAA  431 bp  431 bp TCC-3′ 10 5′-ATA TTT AGT TAG TAT GCA TA-3′ 211 5′- 1021 bp none AAGGAAATCGACGACATCATA-3′ 125′-ATATTTAGTTAGTATGCATA-3′ 3 13 5′-  471 bp  471 bpAAGGAAATCGACGACATCATA-3′ 14 5′-ATA TGG GGT GTC TGT AAA TGT G-3′ 4 155′-ATG TGC GCC GAT TCA ACA 1019 bp none A-3′ 16 5′-AAT CGG GTA TAT ATTGCA CA-3′ 5 17 5′-ATG TGC GCC GAT TCA ACA  993 bp none A-3′ 18 5′-ATATTT AGT TAG TAT GCA TA-3′ 6 19 5′-ATG TGC GCC GAT TCA ACA  443 bp  443bp A-3′ 20 5′-ATA TGG GGT GTC TGT AAA TGT G-3′ 7 21 5′-CAA ACT ATG AAGACC CAA  457 bp  457 bp TCC-3′ 22 5′-AAT CGG GTA TAT ATT GCA CA-3′ 8 235′- 1047 bp 1047 bp AAGGAAATCGACGACATCATA-3′ 245′-AATCGGGTATATATTGCACA-3′

DNA fragments of about 400 to 500 bp of the IHHNV coat protein gene insense and anti-sense orientations for vector construction were used todevelop plasmid constructs for transfer into shrimp.

Example 24 Detection Methods for TSV and IHHNV

Detection of TSV using sequence information from the cDNA segment of theTSV genome and the reverse transcription-polymerase chain reaction(RT-PCR) assay has been developed (Nunan et al., “Reverse TranscriptionPolymerase Chain Reaction (RT-PCR) Used for the Detection of TauraSyndrome Virus (TSV) in Experimentally Infected Shrimp,” Dis. Aquatic.Org. 34:87-91 (1998), which is hereby incorporated by reference in itsentirety) and used widely for monitoring TSV infection in farmedshrimps.

Detection and quantification of IHHNV using real-time polymerase chainreaction (PCR) has been developed (Tang et al., “Detection andQuantification of Infectious Hypodermal and Hematopoietic Necrosis Virusin Penaeid Shrimp by Real-Time PCR,” Dis. Aquat. Org. 44(2):79-85(2001); Dhar et al., “Quantitative Assay for Measuring the TauraSyndrome Virus and Yellow Head Virus Load in Shrimp by Real-Time RT-PCRUsing SYBR Green Chemistry,” J. Virol. Methods. 104(1):69-82 (2002),which are hereby incorporated by reference in their entirety) and isconsidered a rapid and highly sensitive method for IHHNV detection inshrimp. Identification of genetic markers as predictors for IHHNVresistance in shrimp have also been reported (Hizer et al., “RAPDMarkers as Predictors of Infectious Hypodermal and HematopoieticNecrosis Virus (IHHNV Resistance in Shrimp (Litopenaeus Stylirostris),”Genome/Natl. Res. Council Canada 45(1):1-7 (2002), which is herebyincorporated by reference in its entirety).

Example 25 Expression Vector Construction

Expression vectors were constructed consisting of the chimeric shrimpβ-actin promoter, a sense (5′→3′) or antisense (3′→5′) oriented fragmentof the TSV-CP target gene, or a reporter gene. The pSV-β-Galactosidasevector (Promega, Madison, Wis.) or pEGFP-N1 (Clontech, Palo Alto,Calif.) were used as the base vectors. A series of vectors asconstructed are shown in FIGS. 6A-C. Using PCR methodology, NcoI andHind III restriction enzyme sites were created at the 5′ end and 3′ end,respectively, of the β-actin promoter of the present invention,β-ActinP2. The SV40 promoter and enhancer of the pSV-β-Galactosidase(β-Gal) vector were excised through restriction enzyme digestion withNcoI and Hind III, and the β-ActinP2 was inserted into the vector toconstruct the expression vector, pβ-ActinP2-β-Gal, shown in FIG. 6A. Inaddition, Hind III and Sal I restriction enzyme sites were added to the471-bp TSV-CP target gene using PCR. The lacZ gene of thepβ-ActinP2-β-Gal vector was replaced with the TSV-CP target gene inantisense orientation by restriction enzyme digestion with Hind III andSalI to produce the expression vector, pβ-ActinP2-TSV-CP-AS (471 bp), asshown in FIG. 6B. A third expression vector, pβ-ActinP2-TSV-CP-S (471bp) was constructed with the TSV-CP target gene in the sense orientationas shown in FIG. 6C. The expression vectors were cloned, purified, andintroduced into shrimp embryos through electroporation andmicroinjection.

A brief description and plasmid map of these and other vector constructsare provided as follows. FIG. 7 shows the pβ-ActinP2-TSV-CP-S vectorwhich contains the 1234-bp promoter region (β-ActinP2) of the shrimpβ-actin gene. The target gene of this vector is the 491-bp Taurasyndrome virus coat protein (TSV-CP) fragment in sense orientation. FIG.8 shows the pβ-ActinP2-TSV-CP-AS vector containing the 1234-bp promoterregion (β-ActinP2) of the shrimp β-actin gene. The target gene of thisvector is the 491-bp TSV-CP fragment in antisense orientation. FIG. 9shows the pβ-ActinP2-β-Gal vector containing the 1234-bp promoter region(β-ActinP2) of the shrimp β-actin gene. This vector contains the 3301-bplac Z gene for β-Gal as the reporter gene. FIG. 10 shows the ActinP2-P26vector containing the 1234-bp promoter region of the shrimp β-actingene. The target gene of this vector is the 791-bp heat shock protein 26(P26) gene from the brine shrimp, Artemia franciscana. FIG. 11 shows thepβ-ActinP3-EGFP vector containing the 893-bp promoter region (β-ActinP3)of the shrimp beta-actin gene. The 718-bp enhanced green fluorescentprotein (EGFP) gene from the jellyfish is the reporter gene in thisvector. FIG. 12 shows the p-ActinP1-EGFP vector containing a 721-bpfragment of the promoter region (ActinP1) of the shrimp actin gene. The718-bp enhanced green fluorescent protein (EGFP) gene from the jellyfishis the reporter gene in this vector.

Example 26 Delivery of Expression Vectors into Shrimp Embryos byElectroporation

Electroporation experiments were carried out with an Electro SquarePorator ECM 830 (BTX). Optimal conditions for obtaining the highesthatching rate of the shrimp eggs were examined by adjusting variableparameters including voltage, electroporation pulse-length, and numberof pulses. In a trial experiment, the Petri Pulser PP35-2P model wasused. Circular plasmid DNA was dissolved in 0.77 M mannitol in a totalvolume of 2 ml at a concentration of 35 μg/ml. Fertilized eggs werede-coated with a buffer containing 32 g NaCl, 0.8 g KCl, 0.36 g NaHCO₃,and 0.28 g NaH₂PO₄ in one liter of distilled water, pH=7.4 or with 0.1mM of 3-amino-1,2,4 triazole (ATA) prior to electroporation. About 400fertilized shrimp eggs were placed in the petri dish (35×10 mm)containing the DNA/mannitol solution. After the electric pulse, the eggswere returned to clean sea water (28° C.) with aeration. The hatchingrate was recorded and compared from each electroporation setting. Theoptimal settings which provided the highest hatching rate of 35% werefound to be: field strength of 40 V/cm; pulse length of 10 us; and 15pulses.

Example 27 Effect of Temperature on the Survival of L. vannamei upon TSVInfection

L. vannamei exposed to TSV exhibited a markedly higher survival ratewhen reared at a temperature of 32° C. than shrimp raised at 26° C.Nineteen out of twenty shrimp survived TSV exposure when raised at watertemperature of 32° C., whereas no survival was observed when TSV-exposedshrimp were raised at 26° C. TSV was detected in all of the shrimpsamples from the 26° C. tank, while most of the shrimp samples from the32° C. tank were TSV negative according to RT-PCR analysis. This studydemonstrated that temperature is a factor that influences the survivalrate of shrimp challenged with TSV. In light of these preliminaryresults, it is hypothesized that the enhanced survival rate of shrimp at32° C. may be due to reduced viability of TSV at that temperature, ormay be due to heat-activated expression of some gene which functions inthe defense mechanism of shrimp. Results from a pilot experiment showedthat heat shock protein 70 (HSP70) gene was detected in all TSV-infectedsamples.

Example 28 Vector Efficiencies in Shrimp

The efficiency of the shrimp pActinP1-EGFP vector was compared to thechicken pCX-EGFP vector and the pCMV-EGFP-N1 vector, as shown in FIG.13. The vectors were introduced into shrimp via intra-muscular injectionand EGFP expression was monitored by spectrofluorometer (excitationwavelength: 488 nm, emission wavelength: 507 nm). The shrimppActinP1-EGFP vector has an EGFP expression level comparable to thepCMV-EGFP-N1 vector in the shrimp system.

The efficiency of the shrimp pβ-ActinP2-β-Gal vector was determinedthrough electroporation and microinjection of A. franciscana embryos. Asshown in FIGS. 14A-B, the vector pβ-ActinP2-β-Gal exhibits higherbeta-gal expression than the control samples.

Transfection of shrimp embryos of L. vannamei via transfection reagentsincluding SuperFect, Effectene, Jet PEI, and Lipofectamine 2000 wereused to facilitate the delivery of β-ActinP2-TSV-CP-AS vector.Transfection efficiency was evaluated by both the hatching rate ofshrimp embryos and transient gene expression detected through RT-PCR.Optimal DNA delivery conditions were examined by exposure of embryos todifferent ratios of foreign DNA and transfection reagents, incombination with electroporation, as well as different embryonic stages(from 10-50 minutes post-fertilization). Significant inhibition wasobserved in embryos exposed to 0.5 μg DNA/SuperFect ratio, where greaterlevels of free DNA were present. As shown in FIG. 15, the highesthatching rates were observed at 0.1 and 0.2 μg of DNA/μl of SuperFect (1μl=3 μg of active SuperFect), in which the number of eggs hatched wascomparable to control. In addition, manipulation of embryos prior to 18minutes post-fertilization resulted in no hatch. Highest hatchabilitywas observed in embryos manipulated at 35 minutes post-fertilization.

The function of the shrimp expression vectors of pβ-ActinP2-TSV-CP-S andpβ-ActinP2-TSV-CP-AS were tested by introducing the vectors into shrimpembryos via microinjection and electroporation. RT-PCR method was usedto verify target gene (TSV-CP) expression in the putative transgenicshrimp at the mysis stage (day 8 after hatching, as shown in FIG. 16 andFIG. 17). Results from these two experiments show that the promoterβ-actin P2 has the ability to drive the expression of foreign gene inshrimp.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

1. An isolated β-actin nucleic acid promoter molecule from shrimp havinga nucleotide sequence comprising one or more (GC)_(n)-rich regions. 2.The isolated nucleic acid promoter molecule according to claim 1,wherein the nucleic acid promoter molecule has a nucleotide sequence ofSEQ ID NO:
 1. 3. A nucleic acid construct comprising: a nucleic acidmolecule encoding a protein; the nucleic acid promoter moleculeaccording to claim 1, wherein the nucleic acid promoter molecule isoperably linked 5′ to the nucleic acid molecule encoding a protein toinduce transcription of the nucleic acid molecule encoding a protein;and a 3′ regulatory region operably linked to the nucleic acid moleculeencoding a protein.
 4. The nucleic acid construct according to claim 3,wherein the nucleic acid molecule encoding a protein has a senseorientation.
 5. The nucleic acid construct according to claim 3, whereinthe nucleic acid molecule encoding a protein has an antisenseorientation.
 6. An expression vector comprising: the nucleic acidconstruct according to claim
 3. 7. A host cell transduced with thenucleic acid construct according to claim
 3. 8. The host cell accordingto claim 7, wherein the cell is selected from the group consisting of abacterial cell, a virus, a yeast cell, an insect cell, and a crustaceancell.
 9. The host cell according to claim 8, wherein the cell is ashrimp cell.
 10. A transgenic animal transformed with the nucleic acidconstruct according to claim
 3. 11. The transgenic animal according toclaim 10, wherein the animal is selected from the group consisting ofmarine fish, crustaceans, shellfish, and insects.
 12. The transgenicanimal according to claim 11, wherein the animal is a shrimp.
 13. Anucleic acid expression cassette comprising: the β-actin promotermolecule according to claim 1; a multiple cloning site positioned in thenucleic acid construct to permit insertion of a nucleic acid moleculeencoding a protein, whereby the nucleic acid molecule is transcribed; anoperable termination segment; and a nucleic acid molecule encoding adetectable marker.
 14. The nucleic acid expression cassette according toclaim 13, wherein the detectable marker is selected from the groupconsisting of green fluorescent protein, enhanced green fluorescentprotein, β-galactosidase, and luciferase.
 15. A method of imparting toan animal resistance against a pathogen comprising: transforming ananimal with the nucleic acid construct according to claim 3, wherein thenucleic acid molecule encoding a protein encodes for resistance to apathogen.
 16. The method according to claim 15, wherein the pathogen isa virus.
 17. The method according to claim 16, wherein the virus isselected from the group consisting of white spot syndrome virus, yellowhead virus, Taura syndrome virus, and infectious hypodermal andhematopoietic necrosis virus.
 18. The method according to claim 17,wherein the virus is Taura syndrome virus.
 19. The method according toclaim 17, wherein the virus is infectious hypodermal and hematopoieticnecrosis virus.
 20. The method according to claim 15, wherein thenucleic acid molecule encodes a viral coat protein or polypeptide. 21.The method according to claim 20, wherein the viral coat protein orpolypeptide is selected from the group consisting of the coat protein orpolypeptide of white spot syndrome virus, the coat protein orpolypeptide of yellow head virus, the coat protein or polypeptide ofTaura syndrome virus, and the coat protein or polypeptide of infectioushypodermal and hematopoietic necrosis virus.
 22. The method according toclaim 21, wherein the viral coat protein or polypeptide is the coatprotein or polypeptide of Taura syndrome virus.
 23. The method accordingto claim 21, wherein the viral coat protein or polypeptide is the coatprotein or polypeptide of infectious hypodermal and hematopoieticnecrosis virus.
 24. The method according to claim 15, wherein the animalis selected from the group consisting of marine fish, crustaceans,shellfish, and insects.
 25. The method according to claim 24, whereinthe animal is a shrimp.
 26. A transgenic animal prepared according tothe method of claim 15, wherein the animal is selected from the groupconsisting of marine fish, crustaceans, shellfish, and insects.
 27. Thetransgenic animal according to claim 26, wherein the animal is shrimp.28. A method of regulating growth of an animal comprising: transformingan animal with the nucleic acid construct according to claim 3, whereinthe nucleic acid molecule encoding a protein encodes a growth regulatingprotein.
 29. The method according to claim 28, wherein the growthregulating protein is a growth hormone.
 30. The method according toclaim 29, wherein the growth hormone is the androgenic hormone.
 31. Themethod according to claim 28, wherein the animal is selected from thegroup consisting of marine fish, crustaceans, shellfish, and insects.32. The method according to claim 31, wherein the animal is a shrimp.33. A transgenic animal prepared according to claim 28, wherein theanimal is selected from the group consisting of marine fish,crustaceans, shellfish, and insects.
 34. The transgenic animal preparedaccording to the method of claim 33, wherein the animal is shrimp.
 35. Amethod of increasing stress tolerance in an animal comprising:transforming an animal with the nucleic acid construct according toclaim 3, wherein the nucleic acid molecule encoding a protein encodes anucleic acid capable of increasing stress tolerance.
 36. The methodaccording to claim 35, wherein the nucleic acid molecule encoding aprotein encodes a heat shock protein.
 37. A transgenic animal producedby the method according to claim
 35. 38. The transgenic animal preparedaccording to claim 37, wherein the animal is selected from the groupconsisting of marine fish, crustaceans, shellfish, and insects.
 39. Thetransgenic animal prepared according to the method of claim 38, whereinthe animal is a shrimp.
 40. The method according to claim 35, whereinthe stress is cold.
 41. The method according to claim 40, wherein thenucleic acid molecule encoding a protein encodes a heat shock protein.42. The method according to claim 41, wherein the heat shock protein isHSP70 or HSP26.
 43. A transgenic animal produced by the method accordingto claim
 41. 44. The transgenic animal according to claim 43, whereinthe animal is selected from the group consisting of marine fish,crustaceans, shellfish, and insects.
 45. A transgenic animal accordingto claim 44, wherein the animal is a shrimp.
 46. An isolated nucleicacid molecule encoding β-actin from shrimp, wherein the nucleic acidmolecule either 1) has a nucleotide sequence of SEQ ID NO: 2; or 2)encodes a protein having SEQ ID NO:
 3. 47. A nucleic acid constructcomprising: the isolated nucleic acid molecule according to claim 46;and 5′ and 3′ regulatory regions, wherein the regulatory regions areoperably linked to the isolated nucleic acid molecule to allowexpression of the nucleic acid molecule.
 48. An expression vectorcomprising: the nucleic acid construct according to claim
 47. 49. A hostcell transduced with the nucleic acid construct according to claim 47.50. An isolated shrimp β-actin protein or polypeptide having an aminoacid sequence of SEQ ID NO:
 3. 51. An isolated actin nucleic acidpromoter molecule from shrimp having a nucleotide sequence comprising(CATA)-rich repeats and (CACA)-rich repeats.
 52. The isolated nucleicacid promoter molecule according to claim 51, wherein the nucleic acidpromoter molecule has a nucleotide sequence of SEQ ID NO:
 4. 53. Anucleic acid construct comprising: a nucleic acid molecule encoding aprotein; the isolated nucleic acid promoter molecule according to claim51, wherein the nucleic acid promoter molecule is operably linked 5′ tothe nucleic acid molecule encoding a protein to induce transcription ofthe nucleic acid molecule; and an operably linked 3′ regulatory region.54. The nucleic acid construct according to claim 53, wherein thenucleic acid molecule encoding a protein has a sense orientation. 55.The nucleic acid construct according to claim 53, wherein the nucleicacid molecule encoding a protein has an antisense orientation.
 56. Anexpression vector comprising: the nucleic acid construct according toclaim
 53. 57. A host cell transduced with the nucleic acid constructaccording to claim
 53. 58. The host cell according to claim 57, whereinthe cell is selected from the group consisting of a bacterial cell, avirus, a yeast cell, an insect cell, and a crustacean cell.
 59. The hostcell according to claim 58, wherein the cell is a shrimp cell.
 60. Atransgenic animal transformed with the nucleic acid construct accordingto claim
 53. 61. The transgenic animal according to claim 60, whereinthe animal is selected from the group consisting of marine fish,crustaceans, shellfish, and insects.
 62. The transgenic animal accordingto claim 61, wherein the animal is a shrimp.
 63. A nucleic acidexpression cassette comprising: the actin promoter molecule according toclaim 51; a multiple cloning site positioned in the nucleic acidconstruct to permit insertion of a nucleic acid molecule encoding aprotein, whereby the nucleic acid molecule is transcribed, an operabletermination segment; and a nucleic acid molecule encoding a detectablemarker.
 64. The nucleic acid expression cassette according to claim 63,wherein the detectable marker is selected from the group consisting ofgreen fluorescent protein, enhanced green fluorescent protein,β-galactosidase, and luciferase.
 65. A method of imparting to an animalresistance against a pathogen comprising: transforming an animal withthe nucleic acid construct according to claim 53, wherein the nucleicacid molecule encoding a protein encodes for resistance to a pathogen.66. The method according to claim 65, wherein the pathogen is a virus.67. The method according to claim 66, wherein the virus is selected fromthe group consisting of white spot syndrome virus, yellow head virus,Taura syndrome virus, and infectious hypodermal and hematopoieticnecrosis virus.
 68. The method according to claim 67, wherein the virusis Taura syndrome virus.
 69. The method according to claim 67, whereinthe virus is infectious hypodermal and hematopoietic necrosis virus. 70.The method according to claim 66, wherein the nucleic acid moleculeencodes a viral coat protein or polypeptide.
 71. The method according toclaim 70, wherein the viral coat protein or polypeptide is selected fromthe group consisting of the coat protein or polypeptide of white spotsyndrome virus, the coat protein or polypeptide of yellow head virus,the coat protein or polypeptide of Taura syndrome virus, and the coatprotein or polypeptide of infectious hypodermal and hematopoieticnecrosis virus.
 72. The method according to claim 71, wherein the viralcoat protein or polypeptide is the coat protein or polypeptide of Taurasyndrome virus.
 73. The method according to claim 71, wherein the viralcoat protein or polypeptide is the coat protein or polypeptide ofinfectious hypodermal and hematopoietic necrosis virus.
 74. The methodaccording to claim 65, wherein the animal is selected from the groupconsisting of marine fish, crustaceans, shellfish, and insects.
 75. Themethod according to claim 74, wherein the animal is a shrimp.
 76. Atransgenic animal prepared according to the method of claim 65, whereinthe animal is selected from the group consisting of marine fish,crustaceans, shellfish, and insects.
 77. The transgenic animal accordingto claim 76, wherein the animal is a shrimp.
 78. A method of regulatinggrowth of an animal comprising: transforming an animal with a nucleicacid construct according to claim 53, wherein the nucleic acid moleculeencoding a protein encodes for a growth regulating protein.
 79. Themethod according to claim 78, wherein growth regulating protein is agrowth hormone.
 80. The method according to claim 79, wherein the growthhormone is the androgenic hormone.
 81. The method according to claim 78,wherein the animal is selected from the group consisting of marine fish,crustaceans, shellfish, and insects.
 82. The method according to claim81, wherein the animal is a shrimp.
 83. A transgenic animal preparedaccording to the method of claim 78, wherein the animal is selected fromthe group consisting of marine fish, crustaceans, shellfish, andinsects.
 84. The transgenic animal according to claim 83, wherein theanimal is shrimp.
 85. A method of increasing stress tolerance in ananimal comprising: transforming an animal with a nucleic acid constructaccording to claim 53, wherein the nucleic acid molecule encoding aprotein encodes for a protein capable of increasing stress tolerance inthe animal.
 86. The method according to claim 85, wherein the nucleicacid encodes a heat shock protein.
 87. A transgenic animal produced bythe method according to claim
 85. 88. The transgenic animal according toclaim 87, wherein the animal is selected from the group consisting ofmarine fish, crustaceans, shellfish, and insects.
 89. The transgenicanimal according to claim 88, wherein the animal is a shrimp.
 90. Themethod according to claim 85, wherein the stress in cold.
 91. The methodaccording to claim 90, wherein the nucleic acid molecule encoding aprotein encodes a heat shock protein.
 92. The method according to claim91, wherein the heat shock protein is HSP70 or HSP26.
 93. A transgenicanimal produced by the method according to claim
 90. 94. The transgenicanimal prepared according to the method of claim 90, wherein the animalis selected from the group consisting of marine fish, crustaceans,shellfish, and insects.
 95. The transgenic animal according to claim 94,wherein the animal is a shrimp.
 96. An isolated nucleic acid moleculeencoding actin from shrimp, wherein the nucleic acid molecule either 1)has a nucleotide sequence of SEQ ID NO: 5; or 2) encodes a proteinhaving SEQ ID NO:
 6. 97. A nucleic acid construct comprising: theisolated nucleic acid molecule according to claim 96; and 5′ and 3′regulatory regions, wherein the regulatory regions are operably linkedto the isolated nucleic acid molecule to allow expression of the nucleicacid molecule.
 98. An expression vector comprising: the nucleic acidconstruct according to claim
 97. 99. A host cell transduced with thenucleic acid construct according to claim
 97. 100. An isolated shrimpactin protein or polypeptide having an amino acid sequence of SEQ ID NO:6.